Peptides and combination of peptides for use in immunotherapy against epithelial ovarian cancer and other cancers

ABSTRACT

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/777,919, filed 31 Jan. 2020, which is a continuation of U.S.application Ser. No. 16/556,549, filed 30 Aug. 2019, now U.S. Pat. No.10,639,331, issued 5 May 2020, which is a continuation of U.S.application Ser. No. 15/813,610, filed 15 Nov. 2017, now U.S. Pat. No.10,463,696, issued 5 Nov. 2019, which is a continuation of U.S.application Ser. No. 15/209,845, filed 14 Jul. 2016, now U.S. Pat. No.9,889,159, issued 13 Feb. 2018, which claims the benefit of U.S.Provisional Application Ser. No. 62/192,670, filed 15 Jul. 2015, andGreat Britain Application No. 1512369.8, filed 15 Jul. 2015, the contentof each of these applications is herein incorporated by reference intheir entirety.

This application also is related to PCT/EP2016/066706 filed 14 Jul.2016, the content of which is incorporated herein by reference in itsentirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.txt)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “2912919-052008_Sequence_Listing_ST25.txt” createdon 17 Jun. 2020, and 100,077 bytes in size) is submitted concurrentlywith the instant application, and the entire contents of the SequenceListing are incorporated herein by reference.

FIELD

The present invention relates to peptides, proteins, nucleic acids andcells for use in immunotherapeutic methods. In particular, the presentinvention relates to the immunotherapy of cancer. The present inventionfurthermore relates to tumor-associated T-cell peptide epitopes, aloneor in combination with other tumor-associated peptides that can forexample serve as active pharmaceutical ingredients of vaccinecompositions that stimulate anti-tumor immune responses, or to stimulateT cells ex vivo and transfer into patients. Peptides bound to moleculesof the major histocompatibility complex (MHC), or peptides as such, canalso be targets of antibodies, soluble T-cell receptors, and otherbinding molecules.

The present invention relates to several novel peptide sequences andtheir variants derived from HLA class I as well as HLA class IImolecules of human tumor cells that can be used in vaccine compositionsfor eliciting anti-tumor immune responses, or as targets for thedevelopment of pharmaceutically/immunologically active compounds andcells.

BACKGROUND OF THE INVENTION

Epithelial ovarian cancer (EOC) remains the leading cause of death fromgynecologic malignancies and the fifth leading cause of cancer relateddeath in the western world, causing an estimated 22,000 new diagnosesand 14,000 deaths in the US in 2014(1). The only available curativetreatment option is complete surgical tumor removal at an early nonmetastatic stage. However, most patients (>70%) are diagnosed with stageIII or IV disease caused by of a lack of specific early symptoms.Despite progress in chemotherapy regimens and the recent approval ofbevacizumab for first line therapy, the majority of patients relapsewithin few months or years after initial treatment (2, 3).

Considering the severe side-effects and expense associated with treatingcancer, there is a need to identify factors that can be used in thetreatment of cancer in general and ovarian cancer in particular. Thereis also a need to identify factors representing biomarkers for cancer ingeneral and ovarian cancer in particular, leading to better diagnosis ofcancer, assessment of prognosis, and prediction of treatment success.

Immunotherapy of cancer represents an option of specific targeting ofcancer cells while minimizing side effects. Cancer immunotherapy makesuse of the existence of tumor associated antigens. The currentclassification of tumor associated antigens (TAAs) comprises thefollowing major groups:

a) Cancer-testis antigens: The first TAAs ever identified that can berecognized by T cells belong to this class, which was originally calledcancer-testis (CT) antigens because of the expression of its members inhistologically different human tumors and, among normal tissues, only inspermatocytes/spermatogonia of testis and, occasionally, in placenta.Since the cells of testis do not express class I and II HLA molecules,these antigens cannot be recognized by T cells in normal tissues and cantherefore be considered as immunologically tumor-specific. Well-knownexamples for CT antigens are the MAGE family members and NY-ESO-1.b) Differentiation antigens: These TAAs are shared between tumors andthe normal tissue from which the tumor arose. Most of the knowndifferentiation antigens are found in melanomas and normal melanocytes.Many of these melanocyte lineage-related proteins are involved inbiosynthesis of melanin and are therefore not tumor specific butnevertheless are widely used for cancer immunotherapy. Examples include,but are not limited to, tyrosinase and Melan-A/MART-1 for melanoma orPSA for prostate cancer.c) Over-expressed TAAs: Genes encoding widely expressed TAAs have beendetected in histologically different types of tumors as well as in manynormal tissues, generally with lower expression levels. It is possiblethat many of the epitopes processed and potentially presented by normaltissues are below the threshold level for T-cell recognition, whiletheir over-expression in tumor cells can trigger an anticancer responseby breaking previously established tolerance. Prominent examples forthis class of TAAs are Her-2/neu, survivin, telomerase, or WT1.d) Tumor-specific antigens: These unique TAAs arise from mutations ofnormal genes (such as β-catenin, CDK4, etc.). Some of these molecularchanges are associated with neoplastic transformation and/orprogression. Tumor-specific antigens are generally able to induce strongimmune responses without bearing the risk for autoimmune reactionsagainst normal tissues. On the other hand, these TAAs are in most casesonly relevant to the exact tumor on which they were identified and areusually not shared between many individual tumors. Tumor-specificity (or-association) of a peptide may also arise if the peptide originates froma tumor- (-associated) exon in case of proteins with tumor-specific(-associated) isoforms.e) TAAs arising from abnormal post-translational modifications: SuchTAAs may arise from proteins which are neither specific noroverexpressed in tumors but nevertheless become tumor associated byposttranslational processes primarily active in tumors. Examples forthis class arise from altered glycosylation patterns leading to novelepitopes in tumors as for MUC1 or events like protein splicing duringdegradation which may or may not be tumor specific.f) Oncoviral proteins: These TAAs are viral proteins that may play acritical role in the oncogenic process and, because they are foreign(not of human origin), they can evoke a T-cell response. Examples ofsuch proteins are the human papilloma type 16 virus proteins, E6 and E7,which are expressed in cervical carcinoma.

Over the last two decades, EOC has been recognized as a highlyimmunogenic tumor, based on diverse clinical findings. Showing frequentimmune cell infiltration EOC was among the first cancers, where adefinitive association of T-cell infiltration and clinical prognosiscould be established. Within these infiltrating T-cell population tumorreactive and antigen specific T-cells have been identified. Tumorresident regulatory T-cells (Tregs) in contrast are negativelycorrelated with clinical outcome. Further, immune stimulatory cytokineshave been shown to induce compelling tumor responses in individualpatients.

The effectiveness of immunotherapeutic approaches for cancer therapy hasbeen illustrated by the recent development and approval of immunecheckpoint inhibitors shown in melanoma treatment. Moreover, antigenspecific peptide vaccination and adoptive T-cell transfer begin to showsuccess in melanoma and other immunogenic tumors, e.g. renal cellcarcinoma. Personalized immunotherapy even has curative potential andstunning results were presented for individual patients.

T-cell based immunotherapy targets peptide epitopes derived fromtumor-associated or tumor-specific proteins, which are presented bymolecules of the major histocompatibility complex (MHC). The antigensthat are recognized by the tumor specific T lymphocytes, that is, theepitopes thereof, can be molecules derived from all protein classes,such as enzymes, receptors, transcription factors, etc. which areexpressed and, as compared to unaltered cells of the same origin,usually up-regulated in cells of the respective tumor.

There are two classes of MHC-molecules, MHC class I and MHC class II.MHC class I molecules are composed of an alpha heavy chain andbeta-2-microglobulin, MHC class II molecules of an alpha and a betachain. Their three-dimensional conformation results in a binding groove,which is used for non-covalent interaction with peptides.

MHC class I molecules can be found on most nucleated cells. They presentpeptides that result from proteolytic cleavage of predominantlyendogenous proteins, defective ribosomal products (DRIPs) and largerpeptides. However, peptides derived from endosomal compartments orexogenous sources are also frequently found on MHC class I molecules.This non-classical way of class I presentation is referred to ascross-presentation in literature (Brossart and Bevan, 1997; Rock et al.,1990). MHC class II molecules can be found predominantly on professionalantigen presenting cells (APCs), and primarily present peptides ofexogenous or transmembrane proteins that are taken up by APCs e.g.during endocytosis, and are subsequently processed.

Complexes of peptide and MHC class I are recognized by CD8-positive Tcells bearing the appropriate T-cell receptor (TCR), whereas complexesof peptide and MHC class II molecules are recognized byCD4-positive-helper-T cells bearing the appropriate TCR. It is wellknown that the TCR, the peptide and the MHC are thereby present in astoichiometric amount of 1:1:1.

CD4-positive helper T cells play an important role in inducing andsustaining effective responses by CD8-positive cytotoxic T cells. Theidentification of CD4-positive T-cell epitopes derived from tumorassociated antigens (TAA) is of great importance for the development ofpharmaceutical products for triggering anti-tumor immune responses(Gnjatic et al., 2003). At the tumor site, T helper cells, support acytotoxic T cell- (CTL-) friendly cytokine milieu (Mortara et al., 2006)and attract effector cells, e.g. CTLs, natural killer (NK) cells,macrophages, and granulocytes (Hwang et al., 2007).

In the absence of inflammation, expression of MHC class II molecules ismainly restricted to cells of the immune system, especially professionalantigen-presenting cells (APC), e.g., monocytes, monocyte-derived cells,macrophages, dendritic cells. In cancer patients, cells of the tumorhave been found to express MHC class II molecules (Dengjel et al.,2006).

Elongated (longer) peptides of the invention can act as MHC class IIactive epitopes.

T-helper cells, activated by MHC class II epitopes, play an importantrole in orchestrating the effector function of CTLs in anti-tumorimmunity. T-helper cell epitopes that trigger a T-helper cell responseof the TH1 type support effector functions of CD8-positive killer Tcells, which include cytotoxic functions directed against tumor cellsdisplaying tumor-associated peptide/MHC complexes on their cellsurfaces. In this way tumor-associated T-helper cell peptide epitopes,alone or in combination with other tumor-associated peptides, can serveas active pharmaceutical ingredients of vaccine compositions thatstimulate anti-tumor immune responses.

It was shown in mammalian animal models, e.g., mice, that even in theabsence of CD8-positive T lymphocytes, CD4-positive T cells aresufficient for inhibiting manifestation of tumors via inhibition ofangiogenesis by secretion of interferon-gamma (IFNγ) (Beatty andPaterson, 2001; Mumberg et al., 1999). There is evidence for CD4 T cellsas direct anti-tumor effectors (Braumuller et al., 2013; Tran et al.,2014).

Since the constitutive expression of HLA class II molecules is usuallylimited to immune cells, the possibility of isolating class II peptidesdirectly from primary tumors was previously not considered possible.However, Dengjel et al. were successful in identifying a number of MHCClass II epitopes directly from tumors (WO 2007/028574, EP 1 760 088B1).

Since both types of response, CD8 and CD4 dependent, contribute jointlyand synergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens recognized by either CD8+T cells (ligand: MHC class I molecule+ peptide epitope) or byCD4-positive T-helper cells (ligand: MHC class II molecule+ peptideepitope) is important in the development of tumor vaccines.

For an MHC class I peptide to trigger (elicit) a cellular immuneresponse, it also must bind to an MHC-molecule. This process isdependent on the allele of the MHC-molecule and specific polymorphismsof the amino acid sequence of the peptide. MHC-class-1-binding peptidesare usually 8-12 amino acid residues in length and usually contain twoconserved residues (“anchors”) in their sequence that interact with thecorresponding binding groove of the MHC-molecule. In this way each MHCallele has a “binding motif” determining which peptides can bindspecifically to the binding groove.

In the MHC class I dependent immune reaction, peptides not only have tobe able to bind to certain MHC class I molecules expressed by tumorcells, they subsequently also have to be recognized by T cells bearingspecific T cell receptors (TCR).

For proteins to be recognized by T-lymphocytes as tumor-specific or-associated antigens, and to be used in a therapy, particularprerequisites must be fulfilled. The antigen should be expressed mainlyby tumor cells and not, or in comparably small amounts, by normalhealthy tissues. In a preferred embodiment, the peptide should beover-presented by tumor cells as compared to normal healthy tissues. Itis furthermore desirable that the respective antigen is not only presentin a type of tumor, but also in high concentrations (i.e. copy numbersof the respective peptide per cell). Tumor-specific and tumor-associatedantigens are often derived from proteins directly involved intransformation of a normal cell to a tumor cell due to their function,e.g. in cell cycle control or suppression of apoptosis. Additionally,downstream targets of the proteins directly causative for atransformation may be up-regulated and thus may be indirectlytumor-associated. Such indirect tumor-associated antigens may also betargets of a vaccination approach (Singh-Jasuja et al., 2004). It isessential that epitopes are present in the amino acid sequence of theantigen, in order to ensure that such a peptide (“immunogenic peptide”),being derived from a tumor associated antigen, leads to an in vitro orin vivo T-cell-response.

Basically, any peptide able to bind an MHC molecule may function as aT-cell epitope. A prerequisite for the induction of an in vitro or invivo T-cell-response is the presence of a T cell having a correspondingTCR and the absence of immunological tolerance for this particularepitope.

Therefore, TAAs are a starting point for the development of a T cellbased therapy including but not limited to tumor vaccines. The methodsfor identifying and characterizing the TAAs are usually based on the useof T-cells that can be isolated from patients or healthy subjects, orthey are based on the generation of differential transcription profilesor differential peptide expression patterns between tumors and normaltissues. However, the identification of genes over-expressed in tumortissues or human tumor cell lines, or selectively expressed in suchtissues or cell lines, does not provide precise information as to theuse of the antigens being transcribed from these genes in an immunetherapy. This is because only an individual subpopulation of epitopes ofthese antigens are suitable for such an application since a T cell witha corresponding TCR has to be present and the immunological tolerancefor this particular epitope needs to be absent or minimal. In a verypreferred embodiment of the invention it is therefore important toselect only those over- or selectively presented peptides against whicha functional and/or a proliferating T cell can be found. Such afunctional T cell is defined as a T cell, which upon stimulation with aspecific antigen can be clonally expanded and is able to executeeffector functions (“effector T cell”).

In case of targeting peptide-MHC by specific TCRs (e.g. soluble TCRs)and antibodies or other binding molecules (scaffolds) according to theinvention, the immunogenicity of the underlying peptides is secondary.In these cases, the presentation is the determining factor.

SUMMARY OF THE INVENTION

In a first aspect of the present invention, the present inventionrelates to a peptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1 to SEQ ID NO: 549 or a variant sequencethereof which is at least 77%, preferably at least 88%, homologous(preferably at least 77% or at least 88% identical) to SEQ ID NO: 1 toSEQ ID NO: 549, wherein said variant binds to MHC and/or induces T cellscross-reacting with said peptide, or a pharmaceutical acceptable saltthereof, wherein said peptide is not the underlying full-lengthpolypeptide.

The present invention further relates to a peptide of the presentinvention comprising a sequence that is selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 549 or a variant thereof, whichis at least 77%, preferably at least 88%, homologous (preferably atleast 77% or at least 88% identical) to SEQ ID NO: 1 to SEQ ID NO: 549,wherein said peptide or variant thereof has an overall length of between8 and 100, preferably between 8 and 30, and most preferred of between 8and 14 amino acids.

The following tables show the peptides according to the presentinvention, their respective SEQ ID NOs, and the prospective source(underlying) genes for these peptides. All peptides in Table 1 and Table2 bind to HLA-A*02. The peptides in Table 2 have been disclosed beforein large listings as results of high-throughput screenings with higherror rates or calculated using algorithms, but have not been associatedwith cancer at all before. The peptides in Table 3 are additionalpeptides that may be useful in combination with the other peptides ofthe invention. The peptides in Table 4 are furthermore useful in thediagnosis and/or treatment of various other malignancies that involve anover-expression or over-presentation of the respective underlyingpolypeptide.

TABLE 1 Peptides according to the present invention; X = S, R or G SEQID No. Sequence Gene HLA binding 1 QFITSTNTF MUC16 A*24:02 2 STETSTVLYMUC16 A*01 3 AHSKITTAM MUC16 B*39:01 4 AVKTETSTSER MUC16 A*31:01 5AVTNVRTSI MUC16 B*13 6 DALTPLVTI MUC16 B*5101 7 DALVLKTV MUC16 B*51 8DPYKATSAV MUC16 B*51 9 EPETTTSFITY MUC16 B*35 10 ERSPVIQTL MUC16 B*39:0111 ETILTFHAF MUC16 A*25 12 EVISSRGTSM MUC16 A*25 13 EVITSSRTTI MUC16A*25 14 EVTSSGRTSI MUC16 A*25 15 FPEKTTHSF MUC16 B*35 16 FPHSEETTTMMUC16 B*35 17 FPHSEITTL MUC16 B*35 18 FQRQGQTAL MUC16 B*15:01 19GDVPRPSSL MUC16 B*08:01 20 GHESHSPAL MUC16 B*39:01 21 GHTTVSTSM MUC16B*39:01 22 GTHSPVTQR MUC16 A*31:01 23 GTSGTPVSK MUC16 A*11 24 HPDPQSPGLMUC16 B*35 25 IPRVFTSSI MUC16 B*51 26 ISDEVVTRL MUC16 C*05 27 ISIGTIPRIMUC16 B*15:17 28 ISKEDVTSI MUC16 B*15:17 29 ITETSAVLY MUC16 A*01 30ITRLPTSSI MUC16 B*15:17 31 KDTAHTEAM MUC16 B*44:02 32 KEDSTALVM MUC16B*40/B*44 33 KEVTSSSSVL MUC16 B*40/B*44/? 34 LPHSEITTL MUC16 B*35 35LTISTHKTI MUC16 B*15:17 36 LTKSEERTI MUC16 B*15:17 37 RDSLYVNGF MUC16B*44:02 38 RETSTSQKI MUC16 B*18:01 39 RSSGVTFSR MUC16 A*31:01 40SAFESHSTV MUC16 B*51 41 SATERSASL MUC16 C*03/? 42 SENSETTAL MUC16B*40/B*44/? 43 SEQRTSPSL MUC16 ? 44 SESPSTIKL MUC16 B*40/? 45 SPAGEAHSLMUC16 B*07/B*56 46 SPAGEAHSLLA MUC16 B*56:01 47 SPHPVSTTF MUC16 B*07:0248 SPHPVTALL MUC16 B*07:02 49 SPLFQRSSL MUC16 B*0702 50 SPQNLRNTL MUC16B*35/B*07:02 51 SPRLNTQGNTAL MUC16 B*07:02 52 SPSEAITRL MUC16 B*07:02 53SPSKAFASL MUC16 B*35/B*07:02 54 SPSSPTPKV MUC16 B*07:02 55 SPSSQAPVLMUC16 B*07:02 56 SQGFSHSQM MUC16 B*15:01 57 SRTEVISSR MUC16 B*27 58SSAVSTTTI MUC16 B*15:17 59 SSPLRVTSL MUC16 n/a 60 STASSSLSK MUC16 A*1161 STQRVTTSM MUC16 B*07? 62 STSQEIHSATK MUC16 A*11 63 SVLADLVTTK MUC16A*03:01 64 SVPDILSTSW MUC16 A*24:02 65 TAGPTTHQF MUC16 C*03 66TEISSSRTSI MUC16 B*49:01 67 TENTGKEKL MUC16 B*40/B*44 68 TETEAIHVF MUC16B*18 69 TEVSRTEVI MUC16 B*49:01 70 TExVLQGLL MUC16 B*40/B*44/? 71TPGGTRQSL MUC16 B*07:02/B*35 72 TPGNRAISL MUC16 B*07:02/B*35 73TPNSRGETSL MUC16 B*07:02 74 TSGPVTEKY MUC16 B*35 75 TSPAGEAHSL MUC16 ?76 VHESHSSVL MUC16 B*39:01 77 VPRSAATTL MUC16 B*07:02/B*35 78 VTSAPGRSIMUC16 B*15:17 79 VTSSSRTSI MUC16 B*15:17 80 YPDPSKASSAM MUC16 B*35 81AAWLRSAAA MMP11 B*55/B*56 82 APAAWLRSAA MMP11 B*55/B*56 83 APAAWLRSAAAMMP11 B*55/B*56 84 LPSPVDAAF MMP11 B*35 85 RGVPSEIDAAF MMP11 B*58 86EAGPPAFYR ESR1 A*66 87 STSSHSLQK ESR1 A*03/A*11 88 APHLHLSA KLK10B*56:01 89 APHLHLSAA KLK10 B*56:01 90 RALAKLLPL KLK10 B*08/A*02 91SAASGARAL KLK10 C*03 92 VLVDQSWVL KLK10 A*02 93 DYLKRFYLY MMP7 A*24 94SETKNANSL MMP7 B*44/B*41/B*40 95 SSDPNAVMY MMP7 A*01 96 YPFDGPGNTL MMP7B*35 97 YPFDGPGNTLAH MMP7 B*35 98 NEIERVFVW EYA2 B*44:02 99 NVGGLIGTPKEYA2 A*03 100 RVKEMYNTY EYA2 A*30/A*32 101 SAPLRVSQL EYA2 ? 102DTDEYVLKY EFHC1 A*01 103 KDSTKTAF EFHC1 B*44 104 SKAPVLTY EFHC1 B*15:03105 AEYTDVLQKI EPS8L1 B*49 106 EYTDVLQKI EPS8L1 A*24 107 RPHLTSDA EPS8L1B*56 108 RPHLTSDAV EPS8L1 B*56 109 RPHLTSDAVA EPS8L1 B*56 110 SAKSIYEQREPS8L1 A*31 111 SPEEGARVY EPS8L1 B*35 112 SQYPVNHLV EPS8L1 B*15 113YPVNHLVTF EPS8L1 B*35 114 AAASAIKVI IDO1 C*12 115 IHDHVNPKAFF IDO1 B*38116 NPKAFFSVL IDO1 B*07 117 NPSVREFVL IDO1 B*35 118 RSYHLQIVTK IDO1A*11/A*03 119 RYMPPAHRNF IDO1 A*24 120 TEFEQYLHF SOX17 B*18/B*44 121VSDASSAVYY SOX17 A*01 122 AEIEADRSY LAMC2 B*44 123 AQKVDTRAK LAMC2 A*03124 HPSAHDVIL LAMC2 B*35:03 125 RIKQKADSL LAMC2 B*08 126 SEGASRSLGLLAMC2 B*37 127 SVDEEGLVLL LAMC2 A*02 128 SVHKITSTF LAMC2 A*25 129TREATQAEI LAMC2 B*39 130 VYFVAPAKF LAMC2 A*24 131 APQSAHAAF SGPL1 B*07132 ETIIIFHSL EYA3 A*25 133 TELLVKAY SGPL1 B*18 134 WQEGRASGTVY SGPL1B*15 135 IRSENFEEL CRABP2 B*39 136 KIAVAAASK CRABP2 A*03 137 NVMLRKIAVCRABP2 B*08 138 RELTNDGELIL CRABP2 B*40/B*44 139 VAAASKPAV CRABP2 ? 140SPNAIFKAL SOX9 B*07 141 SSKNKPHVKR SOX9 A*31 142 TPASAGHVW SOX9 B*07 143YTDHQNSSSY SOX9 A*01 144 AEVLLPRL MSLN B*40 145 AVLPLTVAEVQK MSLN A*03146 LPTARPLL MSLN B*07 147 RVRELAVAL MSLN A*02 148 NLPIFLPRV MLPH A*02149 RVHPEEQGW MLPH B*58 150 TVKPSGKPR MLPH A*31 151 YYEHVKARF MLPH A*24152 AARPAGATL ERBB2 B*07 153 MPNPEGRYTF ERBB2 B*35 154 FYIKTSTTV CRABP2A*24 155 RTTEINFKV CRABP2 A*02 156 YIKTSTTV CRABP2 B*08 157 GQAAQGPTIDDR1 B*15 158 HRFLAEDAL DDR1 B*39:01 159 EEVARFYAA FOLR1 B*45 160NPNEEVARF FOLR1 B*35 161 NPNEEVARFY FOLR1 B*35 162 KSQTLLGK ULK1A*11/A*03 163 DELISKSF YPEL1 B*18 164 HDELISKSF YPEL1 B*35 165 GRAYLFNSVYPEL1 B*27 166 YLFNSVVNV YPEL1 A*02 167 APDNRPAL MUC1 B*07/B*35 168HHSDTPTTL MUC1 B*38/B*39 169 HPMSEYPTY MUC1 B*35 170 LQRDISEM MUC1 B*51171 LQRDISEMF MUC1 B*51 172 AIAEIGNQL MMP9 A*02 173 DVAQVTGALR MMP9 A*68174 SEDLPRAVI MMP9 B*49/B*40 175 APDAKSFVL LGALS1 B*35 176 EVAPDAKSFLGALS1 A*25 177 FPFQPGSVAEV LGALS1 B*35 178 GEVAPDAKSFVL LGALS1 B*40 179LPDGYEFKF LGALS1 B*35

TABLE 2 Additional peptides according to the present invention, X = S, Ror G SEQ ID MHC No. Sequence class Gene 180 DKAFTAATTEVSR II MUC16 181ELGPYTLDRNSLYVN II MUC16 182 ELGPYTLDRNSLYVNG II MUC16 183FDKAFTAATTEVSR II MUC16 184 GPYTLDRNSLYVN II MUC16 185 LGPYTLDRDSLYVN IIMUC16 186 LGPYTLDRNSLYVN II MUC16 187 LGPYTLDRNSLYVNG II MUC16 188STETITRLSTFPFVTG II MUC16 189 ELQWEQAQDYLKR II MMP7 190 ELQWEQAQDYLKRFII MMP7 191 GINFLYAATHELGHS II MMP7 192 LQWEQAQDYLKR II MMP7 193LQWEQAQDYLKRF II MMP7 194 SELQWEQAQDYLKR II MMP7 195 SELQWEQAQDYLKRF IIMMP7 196 VPYNILTPYPGPR II EPS8L1 197 YVPYNILTPYPGPR II EPS8L1 198GNWKIIRSENFEEL II CRABP2 199 GNWKIIRSENFEELLK II CRABP2 200NWKIIRSENFEEL II CRABP2 201 PNFSGNWKIIRSENF II CRABP2 202VMLRKIAVAAASKPA II CRABP2 203 WKIIRSENFEEL II CRABP2 204 LQRYSSDPTGALTII EGFR 205 NPTTYQMDVNPEGK II EGFR 206 NPTTYQMDVNPEGKY II EGFR 207DDGGQFVVTTNPVNNDG II CDH1 208 DKEGKVFYSITGQGADTPP II CDH1 209DKEGKVFYSITGQGADTPPV II CDH1 210 DKNMFTINRNTGVI II CDH1 211DKNMFTINRNTGVIS II CDH1 212 DPELPDKNMFTINRNTG II CDH1 213DPELPDKNMFTINRNTGVI II CDH1 214 DPELPDKNMFTINRNTGVIS II CDH1 215DPELPDKNMFTINRNTGVISV II CDH1 216 DPELPDKNMFTINRNTGVISV II CDH1 V 217DPELPDKNMFTINRNTGVISV II CDH1 VT 218 DVNTYNAAIAYTILS II CDH1 219DVNTYNAAIAYTILSQ II CDH1 220 EGKVFYSITGQGADT II CDH1 221EGKVFYSITGQGADTPP II CDH1 222 EGKVFYSITGQGADTPPV II CDH1 223ELPDKNMFTINRNTGVIS II CDH1 224 GGQFVVTTNPVNN II CDH1 225 GKVFYSITGQGADTII CDH1 226 GPFPKNLVQIKSNKDK II CDH1 227 GPFPKNLVQIKSNKDKE II CDH1 228GPFPKNLVQIKSNKDKEGK II CDH1 229 KNMFTINRNTGVI II CDH1 230 KNMFTINRNTGVISII CDH1 231 LPDKNMFTINRNTG II CDH1 232 LPDKNMFTINRNTGVI II CDH1 233LPDKNMFTINRNTGVIS II CDH1 234 PELPDKNMFTINRNTGVI II CDH1 235PELPDKNMFTINRNTGVIS II CDH1 236 QDPELPDKNMFTINRNTGVIS II CDH1 237SQDPELPDKNMFTINRNTGVI II CDH1 S 238 SQDPELPDKNMFTINRNTGVI II CDH1 SVVT239 SVPRYLPRPANPDE II CDH1 240 TDGVITVKRPLRFHNPQ II CDH1 241TRAELDREDFEHVK II CDH1 242 VPRYLPRPANPDE II CDH1 243 ALEFRALEPQGLL IIAGRN 244 ALEFRALEPQGLLL II AGRN 245 DTRIFFVNPAPPY II AGRN 246DTRIFFVNPAPPYL II AGRN 247 DTRIFFVNPAPPYLW II AGRN 248 DTRIFFVNPAPPYLWPII AGRN 249 DTRIFFVNPAPPYLWPA II AGRN 250 EFRALEPQGLLL II AGRN 251GAPVPAFEGRSFLAFPTL II AGRN 252 GDTRIFFVNPAPPYLWP II AGRN 253GDTRIFFVNPAPPYLWPA II AGRN 254 IVDVHFDPTTAFRAPD II AGRN 255KVRVWRYLKGKDLVAR II AGRN 256 LALEFRALEPQGLLL II AGRN 257 LEFRALEPQGLLLII AGRN 258 SGPFLADFNGFSH II AGRN 259 TGDTRIFFVNPAPPYLWPA II AGRN 260TRIFFVNPAPPYL II AGRN 261 VDVHFDPTTAFRAPD II AGRN 262 VDVHFDPTTAFRAPDVII AGRN 263 VRVWRYLKGKDLVAR II AGRN 264 APVPAFEGRSFLAFPT II AGRN 265APVPAFEGRSFLAFPTL II AGRN 266 ALRGLLPVLGQPIIR II MSLN 267DLPGRFVAESAEVLLP II MSLN 268 DLPGRFVAESAEVLLPR II MSLN 269 GQPIIRSIPQGIVII MSLN 270 GQPIIRSIPQGIVA II MSLN 271 LGQPIIRSIPQGIVA II MSLN 272LPAALACWGVRGSL II MSLN 273 LPGRFVAESAEVLL II MSLN 274 LPGRFVAESAEVLLP IIMSLN 275 LPGRFVAESAEVLLPR II MSLN 276 LRGLLPVLGQPIIR II MSLN 277PGRFVAESAEVLLPR II MSLN 278 PGRFVAESAEVLLPRL II MSLN 279 QPIIRSIPQGIVAII MSLN 280 RGLLPVLGQPIIR II MSLN 281 SRTLAGETGQEAAPL II MSLN 282STERVRELAVALAQK II MSLN 283 TDAVLPLTVAEVQ II MSLN 284 VAEVQKLLGPHVEG IIMSLN 285 VAEVQKLLGPHVEGLK II MSLN 286 VLGQPIIRSIPQGIVA II MSLN 287VRGSLLSEADVRALG II MSLN 288 VRGSLLSEADVRALGG II MSLN 289 LPAALACWGVRGSLLII MSLN 290 AIKVLRENTSPKANKE II ERBB2 291 DPSPLQRYSEDPTVPLPS II ERBB2292 DPSPLQRYSEDPTVPLPSE II ERBB2 293 ELVSEFSRMARD II ERBB2 294ELVSEFSRMARDPQ II ERBB2 295 IPVAIKVLRENTSPKANKE II ERBB2 296RRLLQETELVEPLTPS II ERBB2 297 SPQPEYVNQPDVRPQPP II ERBB2 298VKPDLSYMPIWKFPDE II ERBB2 299 ASGMRYLATLNFVHR II DDR1 300IASGMRYLATLNFVHR II DDR1 301 KEVKIMSRLKDPN II DDR1 302 LNQFLSAHQLEDK IIDDR1 303 NPAYRLLLATYARPP II DDR1 304 NPAYRLLLATYARPPR II DDR1 305SNPAYRLLLATYARPP II DDR1 306 SNPAYRLLLATYARPPR II DDR1 307DPSTDYYQELQRDISE II MUC1 308 VETQFNQYKTEAASR II MUC1 309GRQVWVYTGASVLGPR II MMP9 310 NQLYLFKDGKYWRFSEG II MMP9 311RQVWVYTGASVLGPR II MMP9 312 SGRQVWVYTGASVLG II MMP9 313 SGRQVWVYTGASVLGPII MMP9 314 SGRQVWVYTGASVLGPR II MMP9 315 VDPRSASEVDRMFPG II MMP9 316GEVAPDAKSFVLN II LGALS1 317 LTVKLPDGYEFKFPNRLNL II LGALS1 318VRGEVAPDAKSFVLN II LGALS1 319 VRGEVAPDAKSFVLNLG II LGALS1

TABLE 3 Additional peptides useful for cancer therapies, X = S, R or GSEQ ID MHC No. Sequence class Gene 320 ATSKIPLAL I MUC16 321 ITSSRTTI IMUC16 322 LNFTITNLQ I MUC16 323 TATSPMVPAS I MUC16 324 TTLPESRPS I MUC16325 VELRVLALP I LRFN4 326 AEDNLIHKF I NLRP2 327 REDLERLGV I NLRP7 328DTKDPAVTEW I TLR7 329 ILISKLLGA I TLR7 330 SESLRTLEF I TLR7 331VLAELVAKL I TLR7 332 INTSILLIF I TLR3 333 ALQPLLHTV I IL17RD 334RLMDNLPQL I IL17RD 335 LIISPTREL I DDX10 336 ADSKVLLF I WDR35 337DSLLEQANNAI I WDR35 338 DYQGIKFVKR I WDR35 339 EVVGYFGRF I WDR35 340KYVKGLISI I WDR35 341 SIGTPLDPK I WDR35 342 TASDKILIV I WDR35 343GVIKVISGF I NOC3L 344 KVKLENKLK I NOC3L 345 SSSEPVHAK I NOC3L 346SSSEPVHAKK I NOC3L 347 LSDQLAQAI I DNASE1 348 LSDIVIEKY I WDR27 349SLDDHVVAV I WDR27 350 SQIDQQNSV I LRIF1 351 STIDPSGTRSK I LRIF1 352VFRDQEPKI I LRIF1 353 VLREKEAAL I LRIF1 354 TRLQQAQAL I POLR2J3 355VAAPEHISY I POLR2J3 356 NSKKKVAL I DDX52 357 QNSKKKVAL I DDX52 358RDNTVHSF I DDX52 359 KQVSEFMTW I RASGEF1B 360 KTKPQSIQR I RASGEF1B 361THIELERL I RASGEF1B 362 IAPKILQL I RASGEF1B 363 DIASVSGRW I BICC1 364KPKQPSKSV I BICC1 365 MPAETIKEL I BICC1 366 SAVKEGTAM I BICC1 367EEEKLQAAF I COMMD10 368 DEFNLQKM I EMC1 369 DEYKVTAF I EMC1 370ETNIGGLNW I EMC1 371 FPQTALVSF I EMC1 372 GEFGKKADGLL I EMC1 373GSMGSFSEK I EMC1 374 IFLIDGVTGRI I EMC1 375 IPPEVQRI I EMC1 376IPYSPDVQI I EMC1 377 QVAPPVLKR I EMC1 378 TEKNVIAAL I EMC1 379 VGKVKFASLI EMC1 380 VPFSHVNI I EMC1 381 VVYQYWNTK I EMC1 382 YPSKQFDVL I EMC1 383AADDSADKV I ZNF217 384 HHKEKQTDV I ZNF217 385 KQTDVAAEV I ZNF217 386KSAFPAQSK I ZNF217 387 NEVVQVHAA I ZNF217 388 SEDLNKHVL I ZNF217 389GETIHIPTM I BCAT1 390 GPKLASRIL I BCAT1 391 GVKKPTKAL I BCAT1 392KEKPDPNNL I BCAT1 393 KVSERYLTM I BCAT1 394 LPVFDKEEL I BCAT1 395LSKLTDIQY I BCAT1 396 DLSNIINKL I WDR12 397 RVWDVESGSLK I WDR12 398SPTTSHVGA I WDR12 399 VEIEYVEKY I WDR12 400 VERNKVKAL I WDR12 401REAVSKEDL I PANK2 402 IMGGNSILHSA I STXBP6 403 KQFEGSTSF I STXBP6 404EEFLRQEHF I OASL 405 ETIPSEIQVF I OASL 406 EVGEALKTVL I DMD 407KLEDLEEQL I DMD 408 LKIQSIAL I DMD 409 MNVLTEWLAAT I DMD 410 AIQDKLFQV ICHCHD6 411 FPNFDKQEL I SMARCAD1 412 GQTKEVLVI I SMARCAD1 413 KLIESTSTM ISMARCAD1 414 KPYQKVGL I SMARCAD1 415 KQESIVLKL I SMARCAD1 416 NANNRLLL ISMARCAD1 417 SEVPNGKEV I SMARCAD1 418 TNNIGSIAR I PANK2 419 DAKGRTVSL IGPX8 420 IIKKKEDL I GPX8 421 DVIDVVQAL I C20orf194 422 EEFKITSF IC20orf194 423 SDFEKTGF I C20orf194 424 DEDRLLVVF I USP34 425 HHSNIPMSL IUSP34 426 LFPSLIKNL I USP34 427 NTNIPIGNK I USP34 428 SDQVADLR I USP34429 THFSFPLRL I USP34 430 TYDSVTDKF I USP34 431 AESLYEIRF I TM9SF1 432DEFLGLTHTY I TM9SF1 547 IITEVITRL I MUC16 548 KMISAIPTL I MUC16 549TYSEKTTLF I MUC16

TABLE 4 Additional peptides useful for cancer therapies, X = S, R or GSEQ ID MHC No. Sequence class Gene 433 ALDFFGNGPPVNY II IFI30 434ALDFFGNGPPVNYKT II IFI30 435 DFFGNGPPVNYK II IFI30 436 DFFGNGPPVNYKT IIIFI30 437 DFFGNGPPVNYKTGN II IFI30 438 DFFGNGPPVNYKTGNL II IFI30 439DFFGNGPPVNYKTGNLY II IFI30 440 LQALDFFGNGPPVNYKTGN II IFI30 441QALDFFGNGPPVNYK II IFI30 442 QPPHEYVPVVVTVNGKP II IFI30 443SPLQALDFFGNGPPVNYKTG II IFI30 444 SPLQALDFFGNGPPVNYKTGN II IFI30 445SPLQALDFFGNGPPVNYKTGNLY II IFI30 446 GPPFSSSQSIPVVPR II GPR64 447LPSSLMNNLPAHDM II GPR64 448 LPSSLMNNLPAHDME II GPR64 449LPSSLMNNLPAHDMEL II GPR64 450 SPIGEIQPLSPQPSAPI II GPR64 451DEVTQPFVIDEKTAEIR II PCDHB5 452 KYPELVLDKALDREER II PCDHB5 453KYPELVLDKALDREERPE II PCDHB5 454 VTQPFVIDEKTAEIR II PCDHB5 455DGRTIVDLEGTPVVSPD II FNDC1 456 DGRTIVDLEGTPVVSPDG II FNDC1 457DKPILSLGGKPLVG II FNDC1 458 GDGRTIVDLEGTPVVSPD II FNDC1 459GDGRTIVDLEGTPVVSPDG II FNDC1 460 GGDGRTIVDLEGTPVVSPD II FNDC1 461GGDGRTIVDLEGTPVVSPDG II FNDC1 462 GRTIVDLEGTPVVSPD II FNDC1 463KVKEYILSYAPALKPF II FNDC1 464 KVKEYILSYAPALKPFG II FNDC1 465LGGDGRTIVDLEGTPVVSPDG II FNDC1 466 RTHEIKKLASESVYV II FNDC1 467VKEYILSYAPALKPF II FNDC1 468 YSKTQYNQVPSEDFERTPQ II CXADR 469AAPNLSRMGAIPVMIP II CXADR 470 AAPNLSRMGAIPVMIPA II CXADR 471APNLSRMGAIPVMIP II CXADR 472 APNLSRMGAIPVMIPA II CXADR 473GYSKTQYNQVPSEDFERTPQ II CXADR 474 SKTQYNQVPSEDFER II CXADR 475SKTQYNQVPSEDFERTP II CXADR 476 SKTQYNQVPSEDFERTPQ II CXADR 477VAAPNLSRMGAIPVMIPA II CXADR 478 VIILYSGDKIYD II CXADR 479YSKTQYNQVPSEDFER II CXADR 480 GHLFALRSLDYE II PCDHB3 481AAEPGYLVTKVVAVDG II PCDHB3 482 AAEPGYLVTKVVAVDGD II PCDHB3 483AAEPGYLVTKVVAVDGDS II PCDHB3 484 AAEPGYLVTKVVAVDGDSG II PCDHB3 485AEPGYLVTKVVAVDG II PCDHB3 486 AEPGYLVTKVVAVDGD II PCDHB3 487AEPGYLVTKVVAVDGDS II PCDHB3 488 EPGYLVTKVVAVDG II PCDHB3 489EPGYLVTKVVAVDGD II PCDHB3 490 EPGYLVTKVVAVDGDS II PCDHB3 491AEPGYLVTKVVAVD II PCDHB3 492 ADSTEFRPNAPVPLVI II CTPS2 493ADSTEFRPNAPVPLVID II CTPS2 494 DADSTEFRPNAPVPLVI II CTPS2 495DADSTEFRPNAPVPLVID II CTPS2 496 DADSTEFRPNAPVPLVIDM II CTPS2 497DADSTEFRPNAPVPLVIDMP II CTPS2 498 DADSTEFRPNAPVPLVIDMPE II CTPS2 499DSTEFRPNAPVPL II CTPS2 500 DSTEFRPNAPVPLV II CTPS2 501 DSTEFRPNAPVPLVIII CTPS2 502 DSTEFRPNAPVPLVID II CTPS2 503 DSTEFRPNAPVPLVIDMP II CTPS2504 DSTEFRPNAPVPLVIDMPE II CTPS2 505 KDADSTEFRPNAPVPLVID II CTPS2 506STEFRPNAPVPL II CTPS2 507 STEFRPNAPVPLVI II CTPS2 508 STEFRPNAPVPLVID IICTPS2 509 STEFRPNAPVPLVIDMP II CTPS2 510 AGDYTIANARKLIDE II RP2 511ETLERLQEL DMD 512 ADITYAIEADSESVK II FAT1 513 DITYAIEADSESVK II FAT1 514KRDNYQIKVVASDHGE II FAT1 515 KRDNYQIKVVASDHGEK II FAT1 516RDESFVIDRQSGRLK II FAT1 517 RDNYQIKVVASDHGE II FAT1 518 SPSELDRDPAYAIVTII FAT1 519 TPPQFSSVKVIHVTSPQ II FAT1 520 VPLPDIQEFPNY II FAT1 521GPQLFHMDPSGTFVQ II PSMA5 522 DKNYFEGTGYARVPTQP II LAMA3 523DKNYFEGTGYARVPTQPH II LAMA3 524 DSKPLYTPSSSFGVS II LAMA3 525IQRQVKEINSLQSDFT II LAMA3 526 KNYFEGTGYARVPT II LAMA3 527KNYFEGTGYARVPTQP II LAMA3 528 KNYFEGTGYARVPTQPH II LAMA3 529SPRVVPNESIPIIPIP II PTPRG 530 SPRVVPNESIPIIPIPD II PTPRG 531SSPRVVPNESIPIIP II PTPRG 532 SSPRVVPNESIPIIPIP II PTPRG 533SSPRVVPNESIPIIPIPD II PTPRG 534 DDKGYTLMHPSLTRPY II CACHD1 535DVGGAGYVVTISHTIHS II CACHD1 536 GAGYVVTISHTIH II CACHD1 537GAGYVVTISHTIHS II CACHD1 538 GGAGYVVTISHTIH II CACHD1 539GGAGYVVTISHTIHS II CACHD1 540 VGGAGYVVTISHTIHS II CACHD1 541MTRTFHDLEGNAVKRDSG II ERMP1 542 RTFHDLEGNAVKR II ERMP1 543RTFHDLEGNAVKRDSG II ERMP1 544 SGTFFPYSSNPANPK II ERMP1 545SGTFFPYSSNPANPKP II ERMP1 546 TRTFHDLEGNAVKR II ERMP1

The present invention furthermore generally relates to the peptidesaccording to the present invention for use in the treatment ofproliferative diseases, such as, for example, ovarian cancer, non-smallcell lung cancer, small cell lung cancer, kidney cancer, brain cancer,colon or rectum cancer, stomach cancer, liver cancer, pancreatic cancer,prostate cancer, leukemia, breast cancer, Merkel cell carcinoma,melanoma, esophageal cancer, urinary bladder cancer, uterine cancer,gallbladder cancer, bile duct cancer and other tumors that show anoverexpression of a protein from which a peptide SEQ ID No. 1 to SEQ IDNo. 319 is derived from.

Particularly preferred are the peptides—alone or incombination—according to the present invention selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 549. More preferred are thepeptides—alone or in combination—selected from the group consisting ofSEQ ID NO: 1 to SEQ ID NO: 319 (see Table 1 and 2), and their uses inthe immunotherapy of ovarian cancer, non-small cell lung cancer, smallcell lung cancer, kidney cancer, brain cancer, colon or rectum cancer,stomach cancer, liver cancer, pancreatic cancer, prostate cancer,leukemia, breast cancer, Merkel cell carcinoma, melanoma, esophagealcancer, urinary bladder cancer, uterine cancer, gallbladder cancer, andbile duct cancer, and preferably ovarian cancer.

Thus, another aspect of the present invention relates to the use of thepeptides according to the present invention for the—preferablycombined—treatment of a proliferative disease selected from the group ofovarian cancer, non-small cell lung cancer, small cell lung cancer,kidney cancer, brain cancer, colon or rectum cancer, stomach cancer,liver cancer, pancreatic cancer, prostate cancer, leukemia, breastcancer, Merkel cell carcinoma, melanoma, esophageal cancer, urinarybladder cancer, uterine cancer, gallbladder cancer, and bile ductcancer.

The present invention furthermore relates to peptides according to thepresent invention that have the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or—in an elongatedform, such as a length-variant—MHC class-II.

The present invention further relates to the peptides according to thepresent invention wherein said peptides (each) consist or consistessentially of an amino acid sequence according to SEQ ID NO: 1 to SEQID NO: 549.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is part of a fusion protein, inparticular fused to the N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii), or fused to (or into thesequence of) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the present invention. The present inventionfurther relates to the nucleic acid according to the present inventionthat is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing and/or expressing a nucleic acid according to the presentinvention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use in thetreatment of diseases and in medicine, in particular in the treatment ofcancer.

The present invention further relates to antibodies that are specificagainst the peptides according to the present invention or complexes ofsaid peptides according to the present invention with MHC, and methodsof making these.

The present invention further relates to T-cell receptors (TCRs), inparticular soluble TCR (sTCRs) and cloned TCRs engineered intoautologous or allogeneic T cells, and methods of making these, as wellas NK cells or other cells bearing said TCR or cross-reacting with saidTCRs.

The antibodies and TCRs are additional embodiments of theimmunotherapeutic use of the peptides according to the invention athand.

The present invention further relates to a host cell comprising anucleic acid according to the present invention or an expression vectoras described before. The present invention further relates to the hostcell according to the present invention that is an antigen presentingcell, and preferably is a dendritic cell.

The present invention further relates to a method for producing apeptide according to the present invention, said method comprisingculturing the host cell according to the present invention, andisolating the peptide from said host cell or its culture medium.

The present invention further relates to said method according to thepresent invention, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell.

The present invention further relates to the method according to thepresent invention, wherein the antigen-presenting cell comprises anexpression vector capable of expressing or expressing said peptidecontaining SEQ ID No. 1 to SEQ ID No.: 549, preferably containing SEQ IDNo. 1 to SEQ ID No. 319, or a variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellselectively recognizes a cell which expresses a polypeptide comprisingan amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as produced according to the present invention.

The present invention further relates to the use of any peptide asdescribed, the nucleic acid according to the present invention, theexpression vector according to the present invention, the cell accordingto the present invention, the activated T lymphocyte, the T cellreceptor or the antibody or other peptide- and/or peptide-MHC-bindingmolecules according to the present invention as a medicament or in themanufacture of a medicament. Preferably, the medicament is activeagainst cancer.

Preferably, said medicament is for a cellular therapy, a vaccine or aprotein based on a soluble TCR or antibody.

The present invention further relates to a use according to the presentinvention, wherein said cancer cells are ovarian cancer, non-small celllung cancer, small cell lung cancer, kidney cancer, brain cancer, colonor rectum cancer, stomach cancer, liver cancer, pancreatic cancer,prostate cancer, leukemia, breast cancer, Merkel cell carcinoma,melanoma, esophageal cancer, urinary bladder cancer, uterine cancer,gallbladder cancer, and bile duct cancer, and preferably ovarian cancercells.

The present invention further relates to biomarkers based on thepeptides according to the present invention, herein called “targets”that can be used in the diagnosis of cancer, preferably ovarian cancerThe marker can be over-presentation of the peptide(s) themselves, orover-expression of the corresponding gene(s). The markers may also beused to predict the probability of success of a treatment, preferably animmunotherapy, and most preferred an immunotherapy targeting the sametarget that is identified by the biomarker. For example, an antibody orsoluble TCR can be used to stain sections of the tumor to detect thepresence of a peptide of interest in complex with MHC.

Optionally the antibody carries a further effector function such as animmune stimulating domain or toxin.

The present invention also relates to the use of these novel targets inthe context of cancer treatment.

Both therapeutic and diagnostic uses against additional cancerousdiseases are disclosed in the following more detailed description of theunderlying expression products (polypeptides) of the peptides accordingto the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A and 1B describe an embodiment as described herein.

FIGS. 2A-2D describe an embodiment as described herein.

FIGS. 3A and 3B describe an embodiment as described herein.

FIGS. 4A-4D describe an embodiment as described herein.

FIGS. 5A-5C describe an embodiment as described herein.

FIG. 6 describes an embodiment as described herein.

FIGS. 7A and 7B describe an embodiment as described herein.

FIG. 8 describes an embodiment as described herein.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

Stimulation of an immune response is dependent upon the presence ofantigens recognized as foreign by the host immune system. The discoveryof the existence of tumor associated antigens has raised the possibilityof using a host's immune system to intervene in tumor growth. Variousmechanisms of harnessing both the humoral and cellular arms of theimmune system are currently being explored for cancer immunotherapy.

Specific elements of the cellular immune response are capable ofspecifically recognizing and destroying tumor cells. The isolation ofT-cells from tumor infiltrating cell populations or from peripheralblood suggests that such cells play an important role in natural immunedefense against cancer. CD8-positive T-cells in particular, whichrecognize class I molecules of the major histocompatibility complex(MHC)-bearing peptides of usually 8 to 10 amino acid residues derivedfrom proteins or defect ribosomal products (DRIPS) located in thecytosol, play an important role in this response. The MHC-molecules ofthe human are also designated as human leukocyte-antigens (HLA).

The present invention further relates to a peptide according to thepresent invention, wherein said peptide is modified and/or includesnon-peptide bonds as described herein below.

The present invention further relates to a peptide according to thepresent invention, wherein said peptide is part of a fusion protein, inparticular fused to the N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii), or fused to (or into thesequence of) an antibody, such as, for example, an antibody that isspecific for dendritic cells, i.e. binds to dendritic cells.

The present invention further relates to a nucleic acid, encoding for apeptide according to the present invention. The present inventionfurther relates to the nucleic acid according to the present inventionthat is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing, expressing, and/or presenting a nucleic acid according tothe present invention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use inmedicine.

The present invention further relates to antibodies as described furtherbelow, and methods of making them. Preferred are antibodies that arespecific for the peptides of the present invention, and/or for thepeptides of the present invention when bound to their MHC. Preferredantibodies can be monoclonal.

The present invention further relates to T-cell receptors (TCR), inparticular soluble TCR (sTCRs) targeting the peptides according to theinvention and/or the peptide—MHC complexes thereof, and methods ofmaking them.

The present invention further relates to antibodies or other bindingmolecules targeting the peptides according to the invention and/or thepeptide-MHC complexes thereof, and methods of making them.

The present invention further relates to a host cell comprising anucleic acid according to the present invention or an expression vectoras described before. The present invention further relates to the hostcell according to the present invention that is an antigen presentingcell. The present invention further relates to the host cell accordingto the present invention, wherein the antigen presenting cell is adendritic cell.

The present invention further relates to a method of producing a peptideaccording to the present invention, said method comprising culturing thehost cell according to the present invention, and isolating the peptidefrom the host cell and/or its culture medium.

The present invention further relates to an in vitro method forproducing activated T-cells, the method comprising contacting in vitro Tcells with antigen loaded human class I or II MHC molecules expressed onthe surface of a suitable antigen-presenting cell for a period of timesufficient to activate said T cells in an antigen specific manner,wherein said antigen is at least one peptide according to the presentinvention.

The present invention further relates to a method, wherein the antigenis loaded onto class I or II MHC molecules expressed on the surface of asuitable antigen-presenting cell by contacting a sufficient amount ofthe antigen with an antigen-presenting cell.

The present invention further relates to the method according to thepresent invention, wherein the antigen-presenting cell comprises anexpression vector capable of expressing said peptide containing SEQ IDNO: 1 to SEQ ID NO: 549, or a variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, which selectivelyrecognize a cell, which aberrantly expresses a polypeptide comprising anamino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as according to the present invention.

The present invention further relates to the use of any peptidedescribed, a nucleic acid according to the present invention, anexpression vector according to the present invention, a cell accordingto the present invention, or an activated T-cell according to thepresent invention as a medicament or in the manufacture of a medicament.

The present invention further relates to a use according to the presentinvention, wherein said medicament is a vaccine, a cell, a cellpopulation, such as, for example, a cell line, sTCRs and monoclonalantibodies.

The present invention further relates to a use according to the presentinvention, wherein the medicament is active against cancer.

The present invention further relates to a use according to the presentinvention, wherein said cancer cells are cells of ovarian cancer.

The present invention further relates to particular marker proteins andbiomarkers based on the peptides according to the present invention thatcan be used in the diagnosis and/or prognosis of ovarian cancer.

Furthermore, the present invention relates to the use of these noveltargets for cancer treatment.

Further, the present invention relates to a method for producing apersonalized anti-cancer vaccine for an individual patient using adatabase (herein designated also as “warehouse”) of pre-screened tumorassociated peptides.

Stimulation of an immune response is dependent upon the presence ofantigens recognized as foreign by the host immune system. The discoveryof the existence of tumor associated antigens has raised the possibilityof using a host's immune system to intervene in tumor growth. Variousmechanisms of harnessing both the humoral and cellular arms of theimmune system are currently being explored for cancer immunotherapy.

Specific elements of the cellular immune response are capable ofspecifically recognizing and destroying tumor cells. The isolation ofT-cells from tumor-infiltrating cell populations or from peripheralblood suggests that such cells play an important role in natural immunedefense against cancer. CD8-positive T-cells in particular, whichrecognize class I molecules of the major histocompatibility complex(MHC)-bearing peptides of usually 8 to 10 amino acid residues derivedfrom proteins or defect ribosomal products (DRIPS) located in thecytosol, play an important role in this response. The MHC-molecules ofthe human are also designated as human leukocyte-antigens (HLA).

Tremendous progress in the field of cancer immunotherapy during the lastyears has led to its wide appreciation as a potentially curativeaddition or alternative to standard chemotherapeutic approaches. Severalpapers demonstrate the importance of HLA presented mutated and wild typetumor associated antigens as valuable tumor rejection antigens.Therefore, large scale identification of HLA presented cancer specifictumor antigens adds another important piece to the puzzle of ourunderstanding how the immune system identifies and recognizes tumorcells.

In the present invention the inventors focused on epithelial ovariancancer (EOC) with the goal to comprehensively characterize theimmunopeptidome of EOC and evaluate the HLA presented antigens for theirusefulness in clinical applications. So far, only few HLA presentedantigens have been identified for EOC and most clinical studies haverelied on predicted or established cancer testis antigens notnecessarily also frequently presented by EOC, a fact that could beconfirmed by our analysis.

The inventors demonstrate a consistent and high expression of HLA classI molecules on ovarian tumor cells in line with previously publisheddata. Furthermore, the inventors show on a single cell level that EOCalso display a strong expression of HLA-DR molecules. This strongexpression was further underlined by our identification of large amountsof MHC class II ligands emanating from ovarian tumors as well as fromhighly enriched tumor cell fractions.

Profiling of the immunopeptidome of 34 ovarian tumors in comparison tomore than 85 benign sources of different origin, revealed severalhundred EOC associated antigens. Among the TOP100 HLA class I EOCantigens not presented on any of the tissues in our benign dataset MUC16was clearly most exceptional. Concerning both the number of HLA ligandsidentified (>80) and the frequency of presentation in the patient cohort(˜80%) this is unprecedented for any other tumor antigen and tumorentity the inventors have investigated so far. Moreover, the inventorscould establish that more than 70% of HLA ligands derived from MUC16 areimmunogenic and able to prime T cells in healthy individuals renderingmucin 16 an unparalleled first-class antigen for EOC immunotherapy.Immunopeptidome profiling further provides a showcase for apparentmechanistic insights into EOC, which are reflected in the HLA ligandomeof both HLA class I and class II ligands. HLA ligands from importantkinases and phosphatases (DDR1, EYA2), transcription factors (SOX9,SOX17), proteins associated with immunosuppression (IDO1, Galectin 1) aswell as established and suspected molecular markers for EOC (MUC1,KLK10, FOLR1) are only a few to mention. Notably for HLA class II,mesothelin an established ligand of MUC16 has been identified as theTOP1 tumor associated antigen. Several studies have demonstrated thepivotal role of the MUC16/MSLN axis for cell invasion and metastasis inEOC as well as in other tumors such as pancreatic cancer ormesothelioma, suggesting that T-cell epitopes of these antigens shouldbe further tested in other malignancies. The inventors could show thatMSLN staining is directly correlated with MUC16 staining and high MSLNexpression forms a negative prognostic factor in EOC.

For the first time several different benign tissues and cell types(PBMCs, bone marrow, liver, kidney, colon, ovary) have been used forthis kind of selective immunopeptidome profiling. Due to restrictions inthe number of different tissues available for investigation theinventors cannot completely exclude that individual antigens might alsobe presented by HLA molecules in other organs. The establishedfunctional relevance of those antigens for EOC and particularly theimmunogenicity of the respective peptides in healthy individualshowever, make a presentation of these antigens in other tissuesunlikely.

The term “T-cell response” means the specific proliferation andactivation of effector functions induced by a peptide in vitro or invivo. For MHC class I restricted cytotoxic T cells, effector functionsmay be lysis of peptide-pulsed, peptide-precursor pulsed or naturallypeptide-presenting target cells, secretion of cytokines, preferablyInterferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion ofeffector molecules, preferably granzymes or perforins induced bypeptide, or degranulation.

The term “peptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thepeptides are preferably 9 amino acids in length, but can be as short as8 amino acids in length, and as long as 10, 11, or 12 and in case of MHCclass II peptides (elongated variants of the peptides of the invention)they can be as long as 15, 16, 17, 18, 19 or 20 amino acids in length.

Furthermore, the term “peptide” shall include salts of a series of aminoacid residues, connected one to the other typically by peptide bondsbetween the alpha-amino and carbonyl groups of the adjacent amino acids.Preferably, the salts are pharmaceutical acceptable salts of thepeptides, such as, for example, the chloride or acetate(trifluoroacetate) salts. It has to be noted that the salts of thepeptides according to the present invention differ substantially fromthe peptides in their state(s) in vivo, as the peptides are not salts invivo.

The term “peptide” shall also include “oligopeptide”. The term“oligopeptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thelength of the oligopeptide is not critical to the invention, as long asthe correct epitope or epitopes are maintained therein. Theoligopeptides are typically less than about 30 amino acid residues inlength, and greater than about 15 amino acids in length.

The term “the peptides of the present invention” shall also include thepeptides consisting of or comprising a peptide as defined aboveaccording to SEQ ID NO: 1 to SEQ ID NO: 549.

The term “polypeptide” designates a series of amino acid residues,connected one to the other typically by peptide bonds between thealpha-amino and carbonyl groups of the adjacent amino acids. The lengthof the polypeptide is not critical to the invention as long as thecorrect epitopes are maintained. In contrast to the terms peptide oroligopeptide, the term polypeptide is meant to refer to moleculescontaining more than about 30 amino acid residues.

A peptide, oligopeptide, protein or polynucleotide coding for such amolecule is “immunogenic” (and thus is an “immunogen” within the presentinvention), if it is capable of inducing an immune response. In the caseof the present invention, immunogenicity is more specifically defined asthe ability to induce a T-cell response. Thus, an “immunogen” would be amolecule that is capable of inducing an immune response, and in the caseof the present invention, a molecule capable of inducing a T-cellresponse. In another aspect, the immunogen can be the peptide, thecomplex of the peptide with MHC, oligopeptide, and/or protein that isused to raise specific antibodies or TCRs against it.

A class I T cell “epitope” requires a short peptide that is bound to aclass I MHC receptor, forming a ternary complex (MHC class I alphachain, beta-2-microglobulin, and peptide) that can be recognized by a Tcell bearing a matching T-cell receptor binding to the MHC/peptidecomplex with appropriate affinity. Peptides binding to MHC class Imolecules are typically 8-14 amino acids in length, and most typically 9amino acids in length.

In humans there are three different genetic loci that encode MHC class Imolecules (the MHC-molecules of the human are also designated humanleukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02,and HLA-B*07 are examples of different MHC class I alleles that can beexpressed from these loci.

TABLE 5 Expression frequencies F of HLA-A*02 and HLA-A*24 and the mostfrequent HLA-DR serotypes. Frequencies are deduced from haplotypefrequencies Gf within the American population adapted from Mori et al.(Mori et al., 1997) employing the Hardy-Weinberg formula F = 1 − (1 −Gf)². Combinations of A*02 or A*24 with certain HLA-DR alleles might beenriched or less frequent than expected from their single frequenciesdue to linkage disequilibrium. For details refer to Chanock et al.(Chanock et al., 2004). Calculated phenotype Allele Population fromallele frequency A*02 Caucasian (North America)  49.1% A*02 AfricanAmerican (North America)  34.1% A*02 Asian American (North America) 43.2% A*02 Latin American (North American)  48.3% DR1 Caucasian (NorthAmerica)  19.4% DR2 Caucasian (North America)  28.2% DR3 Caucasian(North America)  20.6% DR4 Caucasian (North America)  30.7% DR5Caucasian (North America)  23.3% DR6 Caucasian (North America)  26.7%DR7 Caucasian (North America)  24.8% DR8 Caucasian (North America)  5.7%DR9 Caucasian (North America)  2.1% DR1 African (North) American 13.20%DR2 African (North) American 29.80% DR3 African (North) American 24.80%DR4 African (North) American 11.10% DR5 African (North) American 31.10%DR6 African (North) American 33.70% DR7 African (North) American 19.20%DR8 African (North) American 12.10% DR9 African (North) American  5.80%DR1 Asian (North) American  6.80% DR2 Asian (North) American 33.80% DR3Asian (North) American  9.20% DR4 Asian (North) American 28.60% DR5Asian (North) American 30.00% DR6 Asian (North) American 25.10% DR7Asian (North) American 13.40% DR8 Asian (North) American 12.70% DR9Asian (North) American 18.60% DR1 Latin (North) American 15.30% DR2Latin (North) American 21.20% DR3 Latin (North) American 15.20% DR4Latin (North) American 36.80% DR5 Latin (North) American 20.00% DR6Latin (North) American 31.10% DR7 Latin (North) American 20.20% DR8Latin (North) American 18.60% DR9 Latin (North) American  2.10% A*24Philippines   65% A*24 Russia Nenets   61% A*24:02 Japan   59% A*24Malaysia   58% A*24:02 Philippines   54% A*24 India   47% A*24 SouthKorea   40% A*24 Sri Lanka   37% A*24 China   32% A*24:02 India   29%A*24 Australia West   22% A*24 USA   22% A*24 Russia Samara   20% A*24South America   20% A*24 Europe   18%

The peptides of the invention, preferably when included into a vaccineof the invention as described herein bind to different HLA types. Avaccine may also include pan-binding MHC class II peptides and peptidesbinding to other alleles, which will be helpful for, personalizedmedicines. Therefore, the vaccine of the invention can be used to treatcancer in patients that are A*02 positive, whereas no selection for MHCclass II allotypes is necessary due to the pan-binding nature of thesepeptides.

In a preferred embodiment, the term “nucleotide sequence” refers to aheteropolymer of deoxyribonucleotides.

The nucleotide sequence coding for a particular peptide, oligopeptide,or polypeptide may be naturally occurring or they may be syntheticallyconstructed. Generally, DNA segments encoding the peptides,polypeptides, and proteins of this invention are assembled from cDNAfragments and short oligonucleotide linkers, or from a series ofoligonucleotides, to provide a synthetic gene that is capable of beingexpressed in a recombinant transcriptional unit comprising regulatoryelements derived from a microbial or viral operon.

As used herein the term “a nucleotide coding for (or encoding) apeptide” refers to a nucleotide sequence coding for the peptideincluding artificial (man-made) start and stop codons compatible for thebiological system the sequence is to be expressed by, for example, adendritic cell or another cell system useful for the production of TCRs.

As used herein, reference to a nucleic acid sequence includes bothsingle stranded and double stranded nucleic acid. Thus, for example forDNA, the specific sequence, unless the context indicates otherwise,refers to the single strand DNA of such sequence, the duplex of suchsequence with its complement (double stranded DNA) and the complement ofsuch sequence.

The term “coding region” refers to that portion of a gene, which eithernaturally or normally codes for the expression product of that gene inits natural genomic environment, i.e., the region coding in vivo for thenative expression product of the gene.

The coding region can be derived from a non-mutated (“normal”), mutatedor altered gene, or can even be derived from a DNA sequence, or gene,wholly synthesized in the laboratory using methods well known to thoseof skill in the art of DNA synthesis.

The term “expression product” means the polypeptide or protein that isthe natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid(s).

The term “fragment”, when referring to a coding sequence, means aportion of DNA comprising less than the complete coding region, whoseexpression product retains essentially the same biological function oractivity as the expression product of the complete coding region.

The term “DNA segment” refers to a DNA polymer, in the form of aseparate fragment or as a component of a larger DNA construct, which hasbeen derived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesegment and its component nucleotide sequences by standard biochemicalmethods, for example, by using a cloning vector. Such segments areprovided in the form of an open reading frame uninterrupted by internalnon-translated sequences, or introns, which are typically present ineukaryotic genes. Sequences of non-translated DNA may be presentdownstream from the open reading frame, where the same do not interferewith manipulation or expression of the coding regions.

The term “primer” means a short nucleic acid sequence that can be pairedwith one strand of DNA and provides a free 3′-OH end at which a DNApolymerase starts synthesis of a deoxyribonucleotide chain.

The term “promoter” means a region of DNA involved in binding of RNApolymerase to initiate transcription.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment, if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polynucleotides, and recombinant or immunogenic polypeptides,disclosed in accordance with the present invention may also be in“purified” form. The term “purified” does not require absolute purity;rather, it is intended as a relative definition, and can includepreparations that are highly purified or preparations that are onlypartially purified, as those terms are understood by those of skill inthe relevant art. For example, individual clones isolated from a cDNAlibrary have been conventionally purified to electrophoretichomogeneity. Purification of starting material or natural material to atleast one order of magnitude, preferably two or three orders, and morepreferably four or five orders of magnitude is expressly contemplated.Furthermore, a claimed polypeptide which has a purity of preferably99.999%, or at least 99.99% or 99.9%; and even desirably 99% by weightor greater is expressly disclosed.

The nucleic acids and polypeptide expression products disclosedaccording to the present invention, as well as expression vectorscontaining such nucleic acids and/or such polypeptides, may be in“enriched form”. As used herein, the term “enriched” means that theconcentration of the material is at least about 2, 5, 10, 100, or 1000times its natural concentration (for example), advantageously 0.01%, byweight, preferably at least about 0.1% by weight. Enriched preparationsof about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. Thesequences, constructs, vectors, clones, and other materials comprisingthe present invention can advantageously be in enriched or isolatedform.

As used herein, the terms “portion”, “segment” and “fragment”, when usedin relation to polypeptides, refer to a continuous sequence of residues,such as amino acid residues, which sequence forms a subset of a largersequence. For example, if a polypeptide were subjected to treatment withany of the common endopeptidases, such as trypsin or chymotrypsin, theoligopeptides resulting from such treatment would represent portions,segments or fragments of the starting polypeptide. When used in relationto polynucleotides, these terms refer to the products produced bytreatment of said polynucleotides with any of the endonucleases.

In accordance with the present invention, the term “percent identity” or“percent identical”, when referring to a sequence, means that a sequenceis compared to a claimed or described sequence after alignment of thesequence to be compared (the “Compared Sequence”) with the described orclaimed sequence (the “Reference Sequence”). The percent identity isthen determined according to the following formula:percent identity=100[1−(C/R)]wherein C is the number of differences between the Reference Sequenceand the Compared Sequence over the length of alignment between theReference Sequence and the Compared Sequence, wherein(i) each base or amino acid in the Reference Sequence that does not havea corresponding aligned base or amino acid in the Compared Sequence and(ii) each gap in the Reference Sequence and(iii) each aligned base or amino acid in the Reference Sequence that isdifferent from an aligned base or amino acid in the Compared Sequence,constitutes a difference and(iiii) the alignment has to start at position 1 of the alignedsequences;and R is the number of bases or amino acids in the Reference Sequenceover the length of the alignment with the Compared Sequence with any gapcreated in the Reference Sequence also being counted as a base or aminoacid.

If an alignment exists between the Compared Sequence and the ReferenceSequence for which the percent identity as calculated above is aboutequal to or greater than a specified minimum Percent Identity then theCompared Sequence has the specified minimum percent identity to theReference Sequence even though alignments may exist in which the hereinabove calculated percent identity is less than the specified percentidentity.

As mentioned above, the present invention thus provides a peptidecomprising a sequence that is selected from the group of consisting ofSEQ ID NO: 1 to SEQ ID NO: 549 or a variant thereof which is 88%homologous to SEQ ID NO: 1 to SEQ ID NO: 549, or a variant thereof thatwill induce T cells cross-reacting with said peptide. The peptides ofthe invention have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or elongated versions of saidpeptides to class II.

In the present invention, the term “homologous” refers to the degree ofidentity (see percent identity above) between sequences of two aminoacid sequences, i.e. peptide or polypeptide sequences. Theaforementioned “homology” is determined by comparing two sequencesaligned under optimal conditions over the sequences to be compared. Sucha sequence homology can be calculated by creating an alignment using,for example, the ClustalW algorithm. Commonly available sequenceanalysis software, more specifically, Vector NTI, GENETYX or other toolsare provided by public databases.

A person skilled in the art will be able to assess, whether T cellsinduced by a variant of a specific peptide will be able to cross-reactwith the peptide itself (Appay et al., 2006; Colombetti et al., 2006;Fong et al., 2001; Zaremba et al., 1997).

By a “variant” of the given amino acid sequence the inventors mean thatthe side chains of, for example, one or two of the amino acid residuesare altered (for example by replacing them with the side chain ofanother naturally occurring amino acid residue or some other side chain)such that the peptide is still able to bind to an HLA molecule insubstantially the same way as a peptide consisting of the given aminoacid sequence in consisting of SEQ ID NO: 1 to SEQ ID NO: 549. Forexample, a peptide may be modified so that it at least maintains, if notimproves, the ability to interact with and bind to the binding groove ofa suitable MHC molecule, such as HLA-A*02 or -DR, and in that way it atleast maintains, if not improves, the ability to bind to the TCR ofactivated T cells.

These T cells can subsequently cross-react with cells and kill cellsthat express a polypeptide that contains the natural amino acid sequenceof the cognate peptide as defined in the aspects of the invention. Ascan be derived from the scientific literature and databases (Rammenseeet al., 1999; Godkin et al., 1997), certain positions of HLA bindingpeptides are typically anchor residues forming a core sequence fittingto the binding motif of the HLA receptor, which is defined by polar,electrophysical, hydrophobic and spatial properties of the polypeptidechains constituting the binding groove. Thus, one skilled in the artwould be able to modify the amino acid sequences set forth in SEQ ID NO:1 to SEQ ID NO 549, by maintaining the known anchor residues, and wouldbe able to determine whether such variants maintain the ability to bindMHC class I or II molecules. The variants of the present inventionretain the ability to bind to the TCR of activated T cells, which cansubsequently cross-react with and kill cells that express a polypeptidecontaining the natural amino acid sequence of the cognate peptide asdefined in the aspects of the invention.

The original (unmodified) peptides as disclosed herein can be modifiedby the substitution of one or more residues at different, possiblyselective, sites within the peptide chain, if not otherwise stated.Preferably those substitutions are located at the end of the amino acidchain. Such substitutions may be of a conservative nature, for example,where one amino acid is replaced by an amino acid of similar structureand characteristics, such as where a hydrophobic amino acid is replacedby another hydrophobic amino acid. Even more conservative would bereplacement of amino acids of the same or similar size and chemicalnature, such as where leucine is replaced by isoleucine. In studies ofsequence variations in families of naturally occurring homologousproteins, certain amino acid substitutions are more often tolerated thanothers, and these are often show correlation with similarities in size,charge, polarity, and hydrophobicity between the original amino acid andits replacement, and such is the basis for defining “conservativesubstitutions.”

Conservative substitutions are herein defined as exchanges within one ofthe following five groups: Group 1-small aliphatic, nonpolar or slightlypolar residues (Ala, Ser, Thr, Pro, Gly); Group 2-polar, negativelycharged residues and their amides (Asp, Asn, Glu, Gln); Group 3-polar,positively charged residues (His, Arg, Lys); Group 4-large, aliphatic,nonpolar residues (Met, Leu, Ile, Val, Cys); and Group 5-large, aromaticresidues (Phe, Tyr, Trp).

Less conservative substitutions might involve the replacement of oneamino acid by another that has similar characteristics but is somewhatdifferent in size, such as replacement of an alanine by an isoleucineresidue. Highly non-conservative replacements might involve substitutingan acidic amino acid for one that is polar, or even for one that isbasic in character. Such “radical” substitutions cannot, however, bedismissed as potentially ineffective since chemical effects are nottotally predictable and radical substitutions might well give rise toserendipitous effects not otherwise predictable from simple chemicalprinciples.

Of course, such substitutions may involve structures other than thecommon L-amino acids. Thus, D-amino acids might be substituted for theL-amino acids commonly found in the antigenic peptides of the inventionand yet still be encompassed by the disclosure herein. In addition,non-standard amino acids (i.e., other than the common naturallyoccurring proteinogenic amino acids) may also be used for substitutionpurposes to produce immunogens and immunogenic polypeptides according tothe present invention.

If substitutions at more than one position are found to result in apeptide with substantially equivalent or greater antigenic activity asdefined below, then combinations of those substitutions will be testedto determine if the combined substitutions result in additive orsynergistic effects on the antigenicity of the peptide. At most, no morethan 4 positions within the peptide would be simultaneously substituted.

A peptide consisting essentially of the amino acid sequence as indicatedherein can have one or two non-anchor amino acids (see below regardingthe anchor motif) exchanged without that the ability to bind to amolecule of the human major histocompatibility complex (MHC) class-I or-II is substantially changed or is negatively affected, when compared tothe non-modified peptide. In another embodiment, in a peptide consistingessentially of the amino acid sequence as indicated herein, one or twoamino acids can be exchanged with their conservative exchange partners(see herein below) without that the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or -II issubstantially changed, or is negatively affected, when compared to thenon-modified peptide.

The amino acid residues that do not substantially contribute tointeractions with the T-cell receptor can be modified by replacementwith other amino acids whose incorporation does not substantially affectT-cell reactivity and does not eliminate binding to the relevant MHC.Thus, apart from the proviso given, the peptide of the invention may beany peptide (by which term the inventors include oligopeptide orpolypeptide), which includes the amino acid sequences or a portion orvariant thereof as given.

Longer (elongated) peptides may also be suitable. It is possible thatMHC class I epitopes, although usually between 8 and 11 amino acidslong, are generated by peptide processing from longer peptides orproteins that include the actual epitope. It is preferred that theresidues that flank the actual epitope are residues that do notsubstantially affect proteolytic cleavage necessary to expose the actualepitope during processing.

The peptides of the invention can be elongated by up to four aminoacids, that is 1, 2, 3 or 4 amino acids can be added to either end inany combination between 4:0 and 0:4. Combinations of the elongationsaccording to the invention can be found in Table 6.

TABLE 6 Combinations of the elongations of peptides of the inventionC-terminus N-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0or 1 or 2 or 3 or 4 N-terminus C-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4

The amino acids for the elongation/extension can be the peptides of theoriginal sequence of the protein or any other amino acid(s). Theelongation can be used to enhance the stability or solubility of thepeptides.

Thus, the epitopes of the present invention may be identical tonaturally occurring tumor-associated or tumor-specific epitopes or mayinclude epitopes that differ by no more than four residues from thereference peptide, as long as they have substantially identicalantigenic activity.

In an alternative embodiment, the peptide is elongated on either or bothsides by more than four amino acids, preferably to a total length of 30amino acids. This may lead to MHC class II binding peptides. Binding toMHC class II can be tested by methods known in the art.

Accordingly, the present invention provides peptides and variants of MHCclass I epitopes, wherein the peptide or variant has an overall lengthof between 8 and 100, preferably between 8 and 30, and most preferredbetween 8 and 14, namely 8, 9, 10, 11, 12, 13, 14 amino acids, in caseof the elongated class II binding peptides the length can also be 15,16, 17, 18, 19, 20, 21 or 22 amino acids.

Of course, the peptide or variant according to the present inventionwill have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class I or II. Binding of a peptide ora variant to a MHC complex may be tested by methods known in the art.

Preferably, when the T cells specific for a peptide according to thepresent invention are tested against the substituted peptides, thepeptide concentration at which the substituted peptides achieve half themaximal increase in lysis relative to background is no more than about 1mM, preferably no more than about 1 μM, more preferably no more thanabout 1 nM, and still more preferably no more than about 100 pM, andmost preferably no more than about 10 pM. It is also preferred that thesubstituted peptide be recognized by T cells from more than oneindividual, at least two, and more preferably three individuals.

In a particularly preferred embodiment of the invention the peptideconsists or consists essentially of an amino acid sequence according toSEQ ID NO: 1 to SEQ ID NO: 549.

“Consisting essentially of” shall mean that a peptide according to thepresent invention, in addition to the sequence according to any of SEQID NO: 1 to SEQ ID NO 549 or a variant thereof contains additional N-and/or C-terminally located stretches of amino acids that are notnecessarily forming part of the peptide that functions as an epitope forMHC molecules epitope.

Nevertheless, these stretches can be important to provide an efficientintroduction of the peptide according to the present invention into thecells. In one embodiment of the present invention, the peptide is partof a fusion protein which comprises, for example, the 80 N-terminalamino acids of the HLA-DR antigen-associated invariant chain (p33, inthe following “Ii”) as derived from the NCBI, GenBank Accession numberX00497. In other fusions, the peptides of the present invention can befused to an antibody as described herein, or a functional part thereof,in particular into a sequence of an antibody, so as to be specificallytargeted by said antibody, or, for example, to or into an antibody thatis specific for dendritic cells as described herein.

In addition, the peptide or variant may be modified further to improvestability and/or binding to MHC molecules in order to elicit a strongerimmune response. Methods for such an optimization of a peptide sequenceare well known in the art and include, for example, the introduction ofreverse peptide bonds or non-peptide bonds.

In a reverse peptide bond amino acid residues are not joined by peptide(—CO—NH—) linkages but the peptide bond is reversed. Such retro-inversopeptidomimetics may be made using methods known in the art, for examplesuch as those described in Meziere et al (1997) (Meziere et al., 1997),incorporated herein by reference. This approach involves makingpseudopeptides containing changes involving the backbone, and not theorientation of side chains. Meziere et al. (Meziere et al., 1997) showthat for MHC binding and T helper cell responses, these pseudopeptidesare useful. Retro-inverse peptides, which contain NH—CO bonds instead ofCO—NH peptide bonds, are much more resistant to proteolysis.

A non-peptide bond is, for example, —CH₂—NH, —CH₂S—, —CH₂CH₂—, —CH═CH—,—COCH₂—, —CH(OH)CH₂—, and —CH₂SO—. U.S. Pat. No. 4,897,445 provides amethod for the solid phase synthesis of non-peptide bonds (—CH₂—NH) inpolypeptide chains which involves polypeptides synthesized by standardprocedures and the non-peptide bond synthesized by reacting an aminoaldehyde and an amino acid in the presence of NaCNBH₃.

Peptides comprising the sequences described above may be synthesizedwith additional chemical groups present at their amino and/or carboxytermini, to enhance the stability, bioavailability, and/or affinity ofthe peptides. For example, hydrophobic groups such as carbobenzoxyl,dansyl, or t-butyloxycarbonyl groups may be added to the peptides' aminotermini. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonylgroup may be placed at the peptides' amino termini. Additionally, thehydrophobic group, t-butyloxycarbonyl, or an amido group may be added tothe peptides' carboxy termini.

Further, the peptides of the invention may be synthesized to alter theirsteric configuration. For example, the D-isomer of one or more of theamino acid residues of the peptide may be used, rather than the usualL-isomer. Still further, at least one of the amino acid residues of thepeptides of the invention may be substituted by one of the well-knownnon-naturally occurring amino acid residues. Alterations such as thesemay serve to increase the stability, bioavailability and/or bindingaction of the peptides of the invention.

Similarly, a peptide or variant of the invention may be modifiedchemically by reacting specific amino acids either before or aftersynthesis of the peptide. Examples for such modifications are well knownin the art and are summarized e.g. in R. Lundblad, Chemical Reagents forProtein Modification, 3rd ed. CRC Press, 2004 (Lundblad, 2004), which isincorporated herein by reference. Chemical modification of amino acidsincludes but is not limited to, modification by acylation, amidination,pyridoxylation of lysine, reductive alkylation, trinitrobenzylation ofamino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amidemodification of carboxyl groups and sulphydryl modification by performicacid oxidation of cysteine to cysteic acid, formation of mercurialderivatives, formation of mixed disulphides with other thiol compounds,reaction with maleimide, carboxymethylation with iodoacetic acid oriodoacetamide and carbamoylation with cyanate at alkaline pH, althoughwithout limitation thereto. In this regard, the skilled person isreferred to Chapter 15 of Current Protocols In Protein Science, Eds.Coligan et al. (John Wiley and Sons NY 1995-2000) (Coligan et al., 1995)for more extensive methodology relating to chemical modification ofproteins.

Briefly, modification of e.g. arginyl residues in proteins is oftenbased on the reaction of vicinal dicarbonyl compounds such asphenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione to form anadduct. Another example is the reaction of methylglyoxal with arginineresidues. Cysteine can be modified without concomitant modification ofother nucleophilic sites such as lysine and histidine. As a result, alarge number of reagents are available for the modification of cysteine.The websites of companies such as Sigma-Aldrich provide information onspecific reagents.

Selective reduction of disulfide bonds in proteins is also common.Disulfide bonds can be formed and oxidized during the heat treatment ofbiopharmaceuticals. Woodward's Reagent K may be used to modify specificglutamic acid residues. N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimidecan be used to form intra-molecular crosslinks between a lysine residueand a glutamic acid residue. For example, diethylpyrocarbonate is areagent for the modification of histidyl residues in proteins. Histidinecan also be modified using 4-hydroxy-2-nonenal. The reaction of lysineresidues and other α-amino groups is, for example, useful in binding ofpeptides to surfaces or the cross-linking of proteins/peptides. Lysineis the site of attachment of poly(ethylene)glycol and the major site ofmodification in the glycosylation of proteins. Methionine residues inproteins can be modified with e.g. iodoacetamide, bromoethylamine, andchloramine T.

Tetranitromethane and N-acetylimidazole can be used for the modificationof tyrosyl residues. Cross-linking via the formation of dityrosine canbe accomplished with hydrogen peroxide/copper ions.

Recent studies on the modification of tryptophan have usedN-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skatole).

Successful modification of therapeutic proteins and peptides with PEG isoften associated with an extension of circulatory half-life whilecross-linking of proteins with glutaraldehyde, polyethylene glycoldiacrylate and formaldehyde is used for the preparation of hydrogels.

Chemical modification of allergens for immunotherapy is often achievedby carbamylation with potassium cyanate.

A peptide or variant, wherein the peptide is modified or includesnon-peptide bonds is a preferred embodiment of the invention. Generally,peptides and variants (at least those containing peptide linkagesbetween amino acid residues) may be synthesized by the Fmoc-polyamidemode of solid-phase peptide synthesis as disclosed by Lukas et al.(Lukas et al., 1981) and by references as cited therein. TemporaryN-amino group protection is afforded by the 9-fluorenylmethyloxycarbonyl(Fmoc) group. Repetitive cleavage of this highly base-labile protectinggroup is done using 20% piperidine in N, N-dimethylformamide. Side-chainfunctionalities may be protected as their butyl ethers (in the case ofserine threonine and tyrosine), butyl esters (in the case of glutamicacid and aspartic acid), butyloxycarbonyl derivative (in the case oflysine and histidine), trityl derivative (in the case of cysteine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). Where glutamine or asparagine are C-terminal residues, use ismade of the 4,4′-dimethoxybenzhydryl group for protection of the sidechain amido functionalities. The solid-phase support is based on apolydimethyl-acrylamide polymer constituted from the three monomersdimethylacrylamide (backbone-monomer), bisacryloylethylene diamine(cross linker) and acryloylsarcosine methyl ester (functionalizingagent). The peptide-to-resin cleavable linked agent used is theacid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All aminoacid derivatives are added as their preformed symmetrical anhydridederivatives with the exception of asparagine and glutamine, which areadded using a reversed N,N-dicyclohexyl-carbodiimide/1hydroxybenzotriazole mediated couplingprocedure. All coupling and deprotection reactions are monitored usingninhydrin, trinitrobenzene sulphonic acid or isotin test procedures.Upon completion of synthesis, peptides are cleaved from the resinsupport with concomitant removal of side-chain protecting groups bytreatment with 95% trifluoroacetic acid containing a 50% scavenger mix.Scavengers commonly used include ethanedithiol, phenol, anisole andwater, the exact choice depending on the constituent amino acids of thepeptide being synthesized. Also a combination of solid phase andsolution phase methodologies for the synthesis of peptides is possible(see, for example, (Bruckdorfer et al., 2004), and the references ascited therein).

Trifluoroacetic acid is removed by evaporation in vacuo, with subsequenttrituration with diethyl ether affording the crude peptide. Anyscavengers present are removed by a simple extraction procedure, whichon lyophilization of the aqueous phase affords the crude peptide free ofscavengers. Reagents for peptide synthesis are generally available frome.g. Calbiochem-Novabiochem (Nottingham, UK).

Purification may be performed by any one, or a combination of,techniques such as re-crystallization, size exclusion chromatography,ion-exchange chromatography, hydrophobic interaction chromatography and(usually) reverse-phase high performance liquid chromatography usinge.g. acetonitrile/water gradient separation.

Analysis of peptides may be carried out using thin layer chromatography,electrophoresis, in particular capillary electrophoresis, solid phaseextraction (CSPE), reverse-phase high performance liquid chromatography,amino-acid analysis after acid hydrolysis and by fast atom bombardment(FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF massspectrometric analysis.

In order to select over-presented peptides, a presentation profile iscalculated showing the median sample presentation as well as replicatevariation. The profile juxtaposes samples of the tumor entity ofinterest to a baseline of normal tissue samples. Each of these profilescan then be consolidated into an over-presentation score by calculatingthe p-value of a Linear Mixed-Effects Model (Pinheiro et al., 2015)adjusting for multiple testing by False Discovery Rate (Benjamini andHochberg, 1995).

For the identification and relative quantitation of HLA ligands by massspectrometry, HLA molecules from shock-frozen tissue samples werepurified and HLA-associated peptides were isolated. The isolatedpeptides were separated and sequences were identified by onlinenano-electrospray-ionization (nanoESI) liquid chromatography-massspectrometry (LC-MS) experiments. The resulting peptide sequences wereverified by comparison of the fragmentation pattern of natural TUMAPsrecorded from ovarian cancer samples with the fragmentation patterns ofcorresponding synthetic reference peptides of identical sequences. Sincethe peptides were directly identified as ligands of HLA molecules ofprimary tumors, these results provide direct evidence for the naturalprocessing and presentation of the identified peptides on primary cancertissue obtained from ovarian cancer patients.

The discovery pipeline XPRESIDENT® v2.1 (see, for example, US2013-0096016, which is hereby incorporated by reference in its entirety)allows the identification and selection of relevant over-presentedpeptide vaccine candidates based on direct relative quantitation ofHLA-restricted peptide levels on cancer tissues in comparison to severaldifferent non-cancerous tissues and organs. This was achieved by thedevelopment of label-free differential quantitation using the acquiredLC-MS data processed by a proprietary data analysis pipeline, combiningalgorithms for sequence identification, spectral clustering, ioncounting, retention time alignment, charge state deconvolution andnormalization.

Presentation levels including error estimates for each peptide andsample were established. Peptides exclusively presented on tumor tissueand peptides over-presented in tumor versus non-cancerous tissues andorgans have been identified.

HLA-peptide complexes from ovarian cancer tissue samples were purifiedand HLA-associated peptides were isolated and analyzed by LC-MS (seeexamples). All TUMAPs contained in the present application wereidentified with this approach on primary ovarian cancer samplesconfirming their presentation on primary ovarian cancer.

TUMAPs identified on multiple ovarian cancer and normal tissues werequantified using ion-counting of label-free LC-MS data. The methodassumes that LC-MS signal areas of a peptide correlate with itsabundance in the sample. All quantitative signals of a peptide invarious LC-MS experiments were normalized based on central tendency,averaged per sample and merged into a bar plot, called presentationprofile. The presentation profile consolidates different analysismethods like protein database search, spectral clustering, charge statedeconvolution (decharging) and retention time alignment andnormalization.

The present invention provides peptides that are useful in treatingcancers/tumors, preferably ovarian cancer that over- or exclusivelypresent the peptides of the invention. These peptides were shown by massspectrometry to be naturally presented by HLA molecules on primary humanovarian cancer samples.

Many of the source gene/proteins (also designated “full-length proteins”or “underlying proteins”) from which the peptides are derived were shownto be highly over-expressed in cancer compared with normaltissues—“normal tissues” in relation to this invention shall mean eitherhealthy ovarian tissue cells or other normal tissue cells, demonstratinga high degree of tumor association of the source genes. Moreover, thepeptides themselves are strongly over-presented on tumor tissue—“tumortissue” in relation to this invention shall mean a sample from a patientsuffering from ovarian cancer, but not on normal tissues.

HLA-bound peptides can be recognized by the immune system, specificallyT lymphocytes. T cells can destroy the cells presenting the recognizedHLA/peptide complex, e.g. ovarian cancer cells presenting the derivedpeptides.

The peptides of the present invention have been shown to be capable ofstimulating T cell responses and/or are over-presented and thus can beused for the production of antibodies and/or TCRs, such as soluble TCRs,according to the present invention. Furthermore, the peptides whencomplexed with the respective MHC can be used for the production ofantibodies and/or TCRs, in particular sTCRs, according to the presentinvention, as well. Respective methods are well known to the person ofskill, and can be found in the respective literature as well. Thus, thepeptides of the present invention are useful for generating an immuneresponse in a patient by which tumor cells can be destroyed. An immuneresponse in a patient can be induced by direct administration of thedescribed peptides or suitable precursor substances (e.g. elongatedpeptides, proteins, or nucleic acids encoding these peptides) to thepatient, ideally in combination with an agent enhancing theimmunogenicity (i.e. an adjuvant). The immune response originating fromsuch a therapeutic vaccination can be expected to be highly specificagainst tumor cells because the target peptides of the present inventionare not presented on normal tissues in comparable copy numbers,preventing the risk of undesired autoimmune reactions against normalcells in the patient.

The present description further relates to T-cell receptors (TCRs)comprising an alpha chain and a beta chain (“alpha/beta TCRs”). Alsoprovided are HAVCR1-001 peptides capable of binding to TCRs andantibodies when presented by an MHC molecule. The present descriptionalso relates to nucleic acids, vectors and host cells for expressingTCRs and peptides of the present description; and methods of using thesame.

The term “T-cell receptor” (abbreviated TCR) refers to a heterodimericmolecule comprising an alpha polypeptide chain (alpha chain) and a betapolypeptide chain (beta chain), wherein the heterodimeric receptor iscapable of binding to a peptide antigen presented by an HLA molecule.The term also includes so-called gamma/delta TCRs.

In one embodiment the description provides a method of producing a TCRas described herein, the method comprising culturing a host cell capableof expressing the TCR under conditions suitable to promote expression ofthe TCR.

The description in another aspect relates to methods according to thedescription, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell or the antigen is loadedonto class I or II MHC tetramers by tetramerizing the antigen/class I orII MHC complex monomers.

The alpha and beta chains of alpha/beta TCR's, and the gamma and deltachains of gamma/delta TCRs, are generally regarded as each having two“domains”, namely variable and constant domains. The variable domainconsists of a concatenation of variable region (V), and joining region(J). The variable domain may also include a leader region (L). Beta anddelta chains may also include a diversity region (D). The alpha and betaconstant domains may also include C-terminal transmembrane (TM) domainsthat anchor the alpha and beta chains to the cell membrane.

With respect to gamma/delta TCRs, the term “TCR gamma variable domain”as used herein refers to the concatenation of the TCR gamma V (TRGV)region without leader region (L), and the TCR gamma J (TRGJ) region, andthe term TCR gamma constant domain refers to the extracellular TRGCregion, or to a C-terminal truncated TRGC sequence. Likewise the term“TCR delta variable domain” refers to the concatenation of the TCR deltaV (TRDV) region without leader region (L) and the TCR delta D/J(TRDD/TRDJ) region, and the term “TCR delta constant domain” refers tothe extracellular TRDC region, or to a C-terminal truncated TRDCsequence.

TCRs of the present description preferably bind to an inventivepeptide-HLA molecule complex with a binding affinity (KD) of about 100μM or less, about 50 μM or less, about 25 μM or less, or about 10 μM orless. More preferred are high affinity TCRs having binding affinities ofabout 1 μM or less, about 100 nM or less, about 50 nM or less, about 25nM or less. Non-limiting examples of preferred binding affinity rangesfor TCRs of the present invention include about 1 nM to about 10 nM;about 10 nM to about 20 nM; about 20 nM to about 30 nM; about 30 nM toabout 40 nM; about 40 nM to about 50 nM; about 50 nM to about 60 nM;about 60 nM to about 70 nM; about 70 nM to about 80 nM; about 80 nM toabout 90 nM; and about 90 nM to about 100 nM.

As used herein in connect with TCRs of the present description,“specific binding” and grammatical variants thereof are used to mean aTCR having a binding affinity (KD) for an HAVCR1-001 peptide-HLAmolecule complex of 100 μM or less.

Alpha/beta heterodimeric TCRs of the present description may have anintroduced disulfide bond between their constant domains. Preferred TCRsof this type include those which have a TRAC constant domain sequenceand a TRBC1 or TRBC2 constant domain sequence except that Thr 48 of TRACand Ser 57 of TRBC1 or TRBC2 are replaced by cysteine residues, the saidcysteines forming a disulfide bond between the TRAC constant domainsequence and the TRBC1 or TRBC2 constant domain sequence of the TCR.

With or without the introduced inter-chain bond mentioned above,alpha/beta heterodimeric TCRs of the present description may have a TRACconstant domain sequence and a TRBC1 or TRBC2 constant domain sequence,and the TRAC constant domain sequence and the TRBC1 or TRBC2 constantdomain sequence of the TCR may be linked by the native disulfide bondbetween Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2.

TCRs of the present description may comprise a detectable label selectedfrom the group consisting of a radionuclide, a fluorophore and biotin.TCRs of the present description may be conjugated to a therapeuticallyactive agent, such as a radionuclide, a chemotherapeutic agent, or atoxin.

In an embodiment, a TCR of the present description having at least onemutation in the alpha chain and/or having at least one mutation in thebeta chain has modified glycosylation compared to the unmutated TCR.

In an embodiment, a TCR comprising at least one mutation in the TCRalpha chain and/or TCR beta chain has a binding affinity for, and/or abinding half-life for, an inventive peptide-HLA molecule complex, whichis at least double that of a TCR comprising the unmutated TCR alphachain and/or unmutated TCR beta chain. Affinity-enhancement oftumor-specific TCRs, and its exploitation, relies on the existence of awindow for optimal TCR affinities. The existence of such a window isbased on observations that TCRs specific for HLA-A2-restricted pathogenshave KD values that are generally about 10-fold lower when compared toTCRs specific for HLA-A2-restricted tumor-associated self-antigens. Itis now known, although tumor antigens have the potential to beimmunogenic, because tumors arise from the individual's own cells onlymutated proteins or proteins with altered translational processing willbe seen as foreign by the immune system. Antigens that are upregulatedor overexpressed (so called self-antigens) will not necessarily induce afunctional immune response against the tumor: T-cells expressing TCRsthat are highly reactive to these antigens will have been negativelyselected within the thymus in a process known as central tolerance,meaning that only T-cells with low-affinity TCRs for self-antigensremain. Therefore, affinity of TCRs or variants of the presentdescription to an inventive peptide can be enhanced by methods wellknown in the art.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising incubating PBMCs from HLA-A*02-negative healthy donors withA2/inventive peptide monomers, incubating the PBMCs withtetramer-phycoerythrin (PE) and isolating the high avidity T-cells byfluorescence activated cell sorting (FACS)-Calibur analysis.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising obtaining a transgenic mouse with the entire human TCRαβ geneloci (1.1 and 0.7 Mb), whose T-cells express a diverse human TCRrepertoire that compensates for mouse TCR deficiency, immunizing themouse with an inventive peptide, incubating PBMCs obtained from thetransgenic mice with tetramer-phycoerythrin (PE), and isolating the highavidity T-cells by fluorescence activated cell sorting (FACS)-Caliburanalysis.

In one aspect, to obtain T-cells expressing TCRs of the presentdescription, nucleic acids encoding TCR-alpha and/or TCR-beta chains ofthe present description are cloned into expression vectors, such asgamma retrovirus or lentivirus. The recombinant viruses are generatedand then tested for functionality, such as antigen specificity andfunctional avidity. An aliquot of the final product is then used totransduce the target T-cell population (generally purified from patientPBMCs), which is expanded before infusion into the patient.

In another aspect, to obtain T-cells expressing TCRs of the presentdescription, TCR RNAs are synthesized by techniques known in the art,e.g., in vitro transcription systems. The in vitro-synthesized TCR RNAsare then introduced into primary CD8+ T-cells obtained from healthydonors by electroporation to re-express tumor specific TCR-alpha and/orTCR-beta chains.

To increase the expression, nucleic acids encoding TCRs of the presentdescription may be operably linked to strong promoters, such asretroviral long terminal repeats (LTRs), cytomegalovirus (CMV), murinestem cell virus (MSCV) U3, phosphoglycerate kinase (PGK), β-actin,ubiquitin, and a simian virus 40 (SV40)/CD43 composite promoter,elongation factor (EF)-1a and the spleen focus-forming virus (SFFV)promoter. In a preferred embodiment, the promoter is heterologous to thenucleic acid being expressed.

In addition to strong promoters, TCR expression cassettes of the presentdescription may contain additional elements that can enhance transgeneexpression, including a central polypurine tract (cPPT), which promotesthe nuclear translocation of lentiviral constructs (Follenzi et al.,2000), and the woodchuck hepatitis virus posttranscriptional regulatoryelement (wPRE), which increases the level of transgene expression byincreasing RNA stability (Zufferey et al., 1999).

The alpha and beta chains of a TCR of the present invention may beencoded by nucleic acids located in separate vectors, or may be encodedby polynucleotides located in the same vector.

Achieving high-level TCR surface expression requires that both theTCR-alpha and TCR-beta chains of the introduced TCR be transcribed athigh levels. To do so, the TCR-alpha and TCR-beta chains of the presentdescription may be cloned into bi-cistronic constructs in a singlevector, which has been shown to be capable of over-coming this obstacle.The use of a viral intraribosomal entry site (IRES) between theTCR-alpha and TCR-beta chains results in the coordinated expression ofboth chains, because the TCR-alpha and TCR-beta chains are generatedfrom a single transcript that is broken into two proteins duringtranslation, ensuring that an equal molar ratio of TCR-alpha andTCR-beta chains are produced. (Schmitt et al. 2009).

Nucleic acids encoding TCRs of the present description may be codonoptimized to increase expression from a host cell. Redundancy in thegenetic code allows some amino acids to be encoded by more than onecodon, but certain codons are less “op-timal” than others because of therelative availability of matching tRNAs as well as other factors(Gustafsson et al., 2004). Modifying the TCR-alpha and TCR-beta genesequences such that each amino acid is encoded by the optimal codon formammalian gene expression, as well as eliminating mRNA instabilitymotifs or cryptic splice sites, has been shown to significantly enhanceTCR-alpha and TCR-beta gene expression (Scholten et al., 2006).

Furthermore, mispairing between the introduced and endogenous TCR chainsmay result in the acquisition of specificities that pose a significantrisk for autoimmunity. For example, the formation of mixed TCR dimersmay reduce the number of CD3 molecules available to form properly pairedTCR complexes, and therefore can significantly decrease the functionalavidity of the cells expressing the introduced TCR (Kuball et al.,2007).

To reduce mispairing, the C-terminus domain of the introduced TCR chainsof the present description may be modified in order to promoteinterchain affinity, while de-creasing the ability of the introducedchains to pair with the endogenous TCR. These strategies may includereplacing the human TCR-alpha and TCR-beta C-terminus domains with theirmurine counterparts (murinized C-terminus domain); generating a secondinterchain disulfide bond in the C-terminus domain by introducing asecond cysteine residue into both the TCR-alpha and TCR-beta chains ofthe introduced TCR (cysteine modification); swapping interactingresidues in the TCR-alpha and TCR-beta chain C-terminus domains(“knob-in-hole”); and fusing the variable domains of the TCR-alpha andTCR-beta chains directly to CD3ζ (CD3ζ fusion). (Schmitt et al. 2009).

In an embodiment, a host cell is engineered to express a TCR of thepresent description. In preferred embodiments, the host cell is a humanT-cell or T-cell progenitor. In some embodiments the T-cell or T-cellprogenitor is obtained from a cancer patient. In other embodiments theT-cell or T-cell progenitor is obtained from a healthy donor. Host cellsof the present description can be allogeneic or autologous with respectto a patient to be treated. In one embodiment, the host is a gamma/deltaT-cell transformed to express an alpha/beta TCR.

A “pharmaceutical composition” is a composition suitable foradministration to a human being in a medical setting. Preferably, apharmaceutical composition is sterile and produced according to GMPguidelines.

The pharmaceutical compositions comprise the peptides either in the freeform or in the form of a pharmaceutically acceptable salt (see alsoabove). As used herein, “a pharmaceutically acceptable salt” refers to aderivative of the disclosed peptides wherein the peptide is modified bymaking acid or base salts of the agent. For example, acid salts areprepared from the free base (typically wherein the neutral form of thedrug has a neutral —NH2 group) involving reaction with a suitable acid.Suitable acids for preparing acid salts include both organic acids,e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, malic acid, malonic acid, succinic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, methane sulfonic acid, ethane sulfonic acid, p-toluenesulfonicacid, salicylic acid, and the like, as well as inorganic acids, e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acidphosphoric acid and the like. Conversely, preparation of basic salts ofacid moieties which may be present on a peptide are prepared using apharmaceutically acceptable base such as sodium hydroxide, potassiumhydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or thelike.

In an especially preferred embodiment, the pharmaceutical compositionscomprise the peptides as salts of acetic acid (acetates), trifluoroacetates or hydrochloric acid (chlorides).

Preferably, the medicament of the present invention is animmunotherapeutics such as a vaccine. It may be administered directlyinto the patient, into the affected organ or systemically i.d., i.m.,s.c., i.p. and i.v., or applied ex vivo to cells derived from thepatient or a human cell line which are subsequently administered to thepatient, or used in vitro to select a subpopulation of immune cellsderived from the patient, which are then re-administered to the patient.If the nucleic acid is administered to cells in vitro, it may be usefulfor the cells to be transfected so as to co-express immune-stimulatingcytokines, such as interleukin-2. The peptide may be substantially pure,or combined with an immune-stimulating adjuvant (see below) or used incombination with immune-stimulatory cytokines, or be administered with asuitable delivery system, for example liposomes. The peptide may also beconjugated to a suitable carrier such as keyhole limpet haemocyanin(KLH) or mannan (see WO 95/18145 and (Longenecker et al., 1993)). Thepeptide may also be tagged, may be a fusion protein, or may be a hybridmolecule. The peptides whose sequence is given in the present inventionare expected to stimulate CD4 or CD8 T cells. However, stimulation ofCD8 T cells is more efficient in the presence of help provided by CD4T-helper cells. Thus, for MHC Class I epitopes that stimulate CD8 Tcells the fusion partner or sections of a hybrid molecule suitablyprovide epitopes, which stimulate CD4-positive T cells. CD4- andCD8-stimulating epitopes are well known in the art and include thoseidentified in the present invention.

In one aspect, the vaccine comprises at least one peptide having theamino acid sequence set forth SEQ ID No. 1 to SEQ ID No. 549, and atleast one additional peptide, preferably two to 50, more preferably twoto 25, even more preferably two to 20 and most preferably two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, fifteen, sixteen, seventeen or eighteen peptides. Thepeptide(s) may be derived from one or more specific TAAs and may bind toMHC class I molecules.

A further aspect of the invention provides a nucleic acid (for example apolynucleotide) encoding a peptide or peptide variant of the invention.The polynucleotide may be, for example, DNA, cDNA, PNA, RNA orcombinations thereof, either single- and/or double-stranded, or nativeor stabilized forms of polynucleotides, such as, for example,polynucleotides with a phosphorothioate backbone and it may or may notcontain introns so long as it codes for the peptide. Of course, onlypeptides that contain naturally occurring amino acid residues joined bynaturally occurring peptide bonds are encodable by a polynucleotide. Astill further aspect of the invention provides an expression vectorcapable of expressing a polypeptide according to the invention.

A variety of methods have been developed to link polynucleotides,especially DNA, to vectors for example via complementary cohesivetermini. For instance, complementary homopolymer tracts can be added tothe DNA segment to be inserted to the vector DNA. The vector and DNAsegment are then joined by hydrogen bonding between the complementaryhomopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. Syntheticlinkers containing a variety of restriction endonuclease sites arecommercially available from a number of sources including InternationalBiotechnologies Inc. New Haven, Conn., USA.

A desirable method of modifying the DNA encoding the polypeptide of theinvention employs the polymerase chain reaction as disclosed by Saiki RK, et al. (Saiki et al., 1988). This method may be used for introducingthe DNA into a suitable vector, for example by engineering in suitablerestriction sites, or it may be used to modify the DNA in other usefulways as is known in the art. If viral vectors are used, pox- oradenovirus vectors are preferred.

The DNA (or in the case of retroviral vectors, RNA) may then beexpressed in a suitable host to produce a polypeptide comprising thepeptide or variant of the invention. Thus, the DNA encoding the peptideor variant of the invention may be used in accordance with knowntechniques, appropriately modified in view of the teachings containedherein, to construct an expression vector, which is then used totransform an appropriate host cell for the expression and production ofthe polypeptide of the invention. Such techniques include thosedisclosed, for example, in U.S. Pat. Nos. 4,440,859, 4,530,901,4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463, 4,757,006,4,766,075, and 4,810,648.

The DNA (or in the case of retroviral vectors, RNA) encoding thepolypeptide constituting the compound of the invention may be joined toa wide variety of other DNA sequences for introduction into anappropriate host. The companion DNA will depend upon the nature of thehost, the manner of the introduction of the DNA into the host, andwhether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognized bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques. Generally, not all of the hosts will be transformedby the vector. Therefore, it will be necessary to select for transformedhost cells. One selection technique involves incorporating into theexpression vector a DNA sequence, with any necessary control elements,that codes for a selectable trait in the transformed cell, such asantibiotic resistance.

Alternatively, the gene for such selectable trait can be on anothervector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the polypeptide, which canthen be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae), filamentous fungi (for example Aspergillus spec.), plantcells, animal cells and insect cells. Preferably, the system can bemammalian cells such as CHO cells available from the ATCC Cell BiologyCollection.

A typical mammalian cell vector plasmid for constitutive expressioncomprises the CMV or SV40 promoter with a suitable poly A tail and aresistance marker, such as neomycin. One example is pSVL available fromPharmacia, Piscataway, N.J., USA. An example of an inducible mammalianexpression vector is pMSG, also available from Pharmacia. Useful yeastplasmid vectors are pRS403-406 and pRS413-416 and are generallyavailable from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA.Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integratingplasmids (Ylps) and incorporate the yeast selectable markers HIS3, TRP1,LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).CMV promoter-based vectors (for example from Sigma-Aldrich) providetransient or stable expression, cytoplasmic expression or secretion, andN-terminal or C-terminal tagging in various combinations of FLAG,3xFLAG, c-myc or MAT. These fusion proteins allow for detection,purification and analysis of recombinant protein. Dual-tagged fusionsprovide flexibility in detection.

The strong human cytomegalovirus (CMV) promoter regulatory region drivesconstitutive protein expression levels as high as 1 mg/L in COS cells.For less potent cell lines, protein levels are typically ˜0.1 mg/L. Thepresence of the SV40 replication origin will result in high levels ofDNA replication in SV40 replication permissive COS cells. CMV vectors,for example, can contain the pMB1 (derivative of pBR322) origin forreplication in bacterial cells, the b-lactamase gene for ampicillinresistance selection in bacteria, hGH polyA, and the f1 origin. Vectorscontaining the pre-pro-trypsin leader (PPT) sequence can direct thesecretion of FLAG fusion proteins into the culture medium forpurification using ANTI-FLAG antibodies, resins, and plates. Othervectors and expression systems are well known in the art for use with avariety of host cells.

In another embodiment two or more peptides or peptide variants of theinvention are encoded and thus expressed in a successive order (similarto “beads on a string” constructs). In doing so, the peptides or peptidevariants may be linked or fused together by stretches of linker aminoacids, such as for example LLLLLL (SEQ ID NO: 559), or may be linkedwithout any additional peptide(s) between them. These constructs canalso be used for cancer therapy, and may induce immune responses bothinvolving MHC I and MHC II.

The present invention also relates to a host cell transformed with apolynucleotide vector construct of the present invention. The host cellcan be either prokaryotic or eukaryotic. Bacterial cells may bepreferred prokaryotic host cells in some circumstances and typically area strain of E. coli such as, for example, the E. coli strains DH5available from Bethesda Research Laboratories Inc., Bethesda, Md., USA,and RR1 available from the American Type Culture Collection (ATCC) ofRockville, Md., USA (No ATCC 31343). Preferred eukaryotic host cellsinclude yeast, insect and mammalian cells, preferably vertebrate cellssuch as those from a mouse, rat, monkey or human fibroblastic and coloncell lines. Yeast host cells include YPH499, YPH500 and YPH501, whichare generally available from Stratagene Cloning Systems, La Jolla,Calif. 92037, USA. Preferred mammalian host cells include Chinesehamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swissmouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, monkeykidney-derived COS-1 cells available from the ATCC as CRL 1650 and 293cells which are human embryonic kidney cells. Preferred insect cells areSf9 cells, which can be transfected with baculovirus expression vectors.An overview regarding the choice of suitable host cells for expressioncan be found in, for example, the textbook of Paulina Balbás and ArgeliaLorence “Methods in Molecular Biology Recombinant Gene Expression,Reviews and Protocols,” Part One, Second Edition, ISBN978-1-58829-262-9, and other literature known to the person of skill.

Transformation of appropriate cell hosts with a DNA construct of thepresent invention is accomplished by well-known methods that typicallydepend on the type of vector used. With regard to transformation ofprokaryotic host cells, see, for example, Cohen et al. (Cohen et al.,1972) and (Green and Sambrook, 2012). Transformation of yeast cells isdescribed in Sherman et al. (Sherman et al., 1986). The method of Beggs(Beggs, 1978) is also useful. With regard to vertebrate cells, reagentsuseful in transfecting such cells, for example calcium phosphate andDEAE-dextran or liposome formulations, are available from StratageneCloning Systems, or Life Technologies Inc., Gaithersburg, Md. 20877,USA. Electroporation is also useful for transforming and/or transfectingcells and is well known in the art for transforming yeast cell,bacterial cells, insect cells and vertebrate cells.

Successfully transformed cells, i.e. cells that contain a DNA constructof the present invention, can be identified by well-known techniquessuch as PCR. Alternatively, the presence of the protein in thesupernatant can be detected using antibodies.

It will be appreciated that certain host cells of the invention areuseful in the preparation of the peptides of the invention, for examplebacterial, yeast and insect cells. However, other host cells may beuseful in certain therapeutic methods. For example, antigen-presentingcells, such as dendritic cells, may usefully be used to express thepeptides of the invention such that they may be loaded into appropriateMHC molecules. Thus, the current invention provides a host cellcomprising a nucleic acid or an expression vector according to theinvention.

In a preferred embodiment the host cell is an antigen presenting cell,in particular a dendritic cell or antigen presenting cell. APCs loadedwith a recombinant fusion protein containing prostatic acid phosphatase(PAP) were approved by the U.S. Food and Drug Administration (FDA) onApr. 29, 2010, to treat asymptomatic or minimally symptomatic metastaticHRPC (Sipuleucel-T) (Rini et al., 2006; Small et al., 2006).

A further aspect of the invention provides a method of producing apeptide or its variant, the method comprising culturing a host cell andisolating the peptide from the host cell or its culture medium.

In another embodiment the peptide, the nucleic acid or the expressionvector of the invention are used in medicine. For example, the peptideor its variant may be prepared for intravenous (i.v.) injection,sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.Preferred methods of peptide injection include s.c., i.d., i.p., i.m.,and i.v. Preferred methods of DNA injection include i.d., i.m., s.c.,i.p. and i.v. Doses of e.g. between 50 μg and 1.5 mg, preferably 125 μgto 500 μg, of peptide or DNA may be given and will depend on therespective peptide or DNA. Dosages of this range were successfully usedin previous trials (Walter et al., 2012).

The polynucleotide used for active vaccination may be substantiallypure, or contained in a suitable vector or delivery system. The nucleicacid may be DNA, cDNA, PNA, RNA or a combination thereof. Methods fordesigning and introducing such a nucleic acid are well known in the art.An overview is provided by e.g. Teufel et al. (Teufel et al., 2005).Polynucleotide vaccines are easy to prepare, but the mode of action ofthese vectors in inducing an immune response is not fully understood.Suitable vectors and delivery systems include viral DNA and/or RNA, suchas systems based on adenovirus, vaccinia virus, retroviruses, herpesvirus, adeno-associated virus or hybrids containing elements of morethan one virus. Non-viral delivery systems include cationic lipids andcationic polymers and are well known in the art of DNA delivery.Physical delivery, such as via a “gene-gun” may also be used. Thepeptide or peptides encoded by the nucleic acid may be a fusion protein,for example with an epitope that stimulates T cells for the respectiveopposite CDR as noted above.

The medicament of the invention may also include one or more adjuvants.Adjuvants are substances that non-specifically enhance or potentiate theimmune response (e.g., immune responses mediated by CD8-positive T cellsand helper-T (TH) cells to an antigen, and would thus be considereduseful in the medicament of the present invention. Suitable adjuvantsinclude, but are not limited to, 1018 ISS, aluminum salts, AMPLIVAX®,AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligandsderived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31, Imiquimod(ALDARA®), resiquimod, ImuFact IMP321, Interleukins as IL-2, IL-13,IL-21, Interferon-alpha or -beta, or pegylated derivatives thereof, ISPatch, ISS, ISCOMATRIX, ISCOMs, JuvImmune®, LipoVac, MALP2, MF59,monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, MontanideISA 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions,OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vector system,poly(lactid co-glycolid) [PLG]-based and dextran microparticles,talactoferrin SRL172, Virosomes and other Virus-like particles, YF-17D,VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, which isderived from saponin, mycobacterial extracts and synthetic bacterialcell wall mimics, and other proprietary adjuvants such as Ribi's Detox,Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred.Several immunological adjuvants (e.g., MF59) specific for dendriticcells and their preparation have been described previously (Allison andKrummel, 1995). Also cytokines may be used. Several cytokines have beendirectly linked to influencing dendritic cell migration to lymphoidtissues (e.g., TNF-), accelerating the maturation of dendritic cellsinto efficient antigen-presenting cells for T-lymphocytes (e.g., GM-CSF,IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specifically incorporatedherein by reference in its entirety) and acting as immunoadjuvants(e.g., IL-12, IL-15, IL-23, IL-7, IFN-alpha. IFN-beta) (Gabrilovich etal., 1996).

CpG immunostimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine setting. Without beingbound by theory, CpG oligonucleotides act by activating the innate(non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9.CpG triggered TLR9 activation enhances antigen-specific humoral andcellular responses to a wide variety of antigens, including peptide orprotein antigens, live or killed viruses, dendritic cell vaccines,autologous cellular vaccines and polysaccharide conjugates in bothprophylactic and therapeutic vaccines. More importantly it enhancesdendritic cell maturation and differentiation, resulting in enhancedactivation of TH1 cells and strong cytotoxic T-lymphocyte (CTL)generation, even in the absence of CD4 T cell help. The TH1 bias inducedby TLR9 stimulation is maintained even in the presence of vaccineadjuvants such as alum or incomplete Freund's adjuvant (IFA) thatnormally promote a TH2 bias. CpG oligonucleotides show even greateradjuvant activity when formulated or co-administered with otheradjuvants or in formulations such as microparticles, nanoparticles,lipid emulsions or similar formulations, which are especially necessaryfor inducing a strong response when the antigen is relatively weak. Theyalso accelerate the immune response and enable the antigen doses to bereduced by approximately two orders of magnitude, with comparableantibody responses to the full-dose vaccine without CpG in someexperiments (Krieg, 2006). U.S. Pat. No. 6,406,705 B1 describes thecombined use of CpG oligonucleotides, non-nucleic acid adjuvants and anantigen to induce an antigen-specific immune response. A CpG TLR9antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen(Berlin, Germany) which is a preferred component of the pharmaceuticalcomposition of the present invention. Other TLR binding molecules suchas RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.

Other examples for useful adjuvants include, but are not limited tochemically modified CpGs (e.g. CpR, Idera), dsRNA analogues such asPoly(I:C) and derivates thereof (e.g. AmpliGen®, Hiltonal®, poly-(ICLC),poly(IC—R), poly(I:C12U), non-CpG bacterial DNA or RNA as well asimmunoactive small molecules and antibodies such as cyclophosphamide,sunitinib, Bevacizumab®, Celebrex, NCX-4016, sildenafil, tadalafil,vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632,pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other antibodiestargeting key structures of the immune system (e.g. anti-CD40,anti-TGFbeta, anti-TNFalpha receptor) and SC58175, which may acttherapeutically and/or as an adjuvant. The amounts and concentrations ofadjuvants and additives useful in the context of the present inventioncan readily be determined by the skilled artisan without undueexperimentation.

Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF,cyclophosphamide, sunitinib, bevacizumab, interferon-alpha, CpGoligonucleotides and derivates, poly-(I:C) and derivates, RNA,sildenafil, and particulate formulations with PLG or Virosomes.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod,resiquimod, and interferon-alpha.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimodand resiquimod. In a preferred embodiment of the pharmaceuticalcomposition according to the invention, the adjuvant iscyclophosphamide, imiquimod or resiquimod. Even more preferred adjuvantsare Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, MontanideISA-51, poly-ICLC (Hiltonal®) and anti-CD40 mAB, or combinationsthereof.

This composition is used for parenteral administration, such assubcutaneous, intradermal, intramuscular or oral administration. Forthis, the peptides and optionally other molecules are dissolved orsuspended in a pharmaceutically acceptable, preferably aqueous carrier.In addition, the composition can contain excipients, such as buffers,binding agents, blasting agents, diluents, flavors, lubricants, etc. Thepeptides can also be administered together with immune stimulatingsubstances, such as cytokines. An extensive listing of excipients thatcan be used in such a composition, can be, for example, taken from A.Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). Thecomposition can be used for a prevention, prophylaxis and/or therapy ofadenomatous or cancerous diseases. Exemplary formulations can be foundin, for example, EP2112253.

It is important to realize that the immune response triggered by thevaccine according to the invention attacks the cancer in differentcell-stages and different stages of development. Furthermore differentcancer associated signaling pathways are attacked. This is an advantageover vaccines that address only one or few targets, which may cause thetumor to easily adapt to the attack (tumor escape). Furthermore, not allindividual tumors express the same pattern of antigens. Therefore, acombination of several tumor-associated peptides ensures that everysingle tumor bears at least some of the targets. The composition isdesigned in such a way that each tumor is expected to express several ofthe antigens and cover several independent pathways necessary for tumorgrowth and maintenance. Thus, the vaccine can easily be used“off-the-shelf” for a larger patient population. This means that apre-selection of patients to be treated with the vaccine can berestricted to HLA typing, does not require any additional biomarkerassessments for antigen expression, but it is still ensured that severaltargets are simultaneously attacked by the induced immune response,which is important for efficacy (Banchereau et al., 2001; Walter et al.,2012).

As used herein, the term “scaffold” refers to a molecule thatspecifically binds to an (e.g. antigenic) determinant. In oneembodiment, a scaffold is able to direct the entity to which it isattached (e.g. a (second) antigen binding moiety) to a target site, forexample to a specific type of tumor cell or tumor stroma bearing theantigenic determinant (e.g. the complex of a peptide with MHC, accordingto the application at hand). In another embodiment a scaffold is able toactivate signaling through its target antigen, for example a T cellreceptor complex antigen. Scaffolds include but are not limited toantibodies and fragments thereof, antigen binding domains of anantibody, comprising an antibody heavy chain variable region and anantibody light chain variable region, binding proteins comprising atleast one ankyrin repeat motif and single domain antigen binding (SDAB)molecules, aptamers, (soluble) TCRs and (modified) cells such asallogenic or autologous T cells. To assess whether a molecule is ascaffold binding to a target, binding assays can be performed.

“Specific” binding means that the scaffold binds the peptide-MHC-complexof interest better than other naturally occurring peptide-MHC-complexes,to an extent that a scaffold armed with an active molecule that is ableto kill a cell bearing the specific target is not able to kill anothercell without the specific target but presenting other peptide-MHCcomplex(es). Binding to other peptide-MHC complexes is irrelevant if thepeptide of the cross-reactive peptide-MHC is not naturally occurring,i.e. not derived from the human HLA-peptidome. Tests to assess targetcell killing are well known in the art. They should be performed usingtarget cells (primary cells or cell lines) with unaltered peptide-MHCpresentation, or cells loaded with peptides such that naturallyoccurring peptide-MHC levels are reached.

Each scaffold can comprise a labeling which provides that the boundscaffold can be detected by determining the presence or absence of asignal provided by the label. For example, the scaffold can be labeledwith a fluorescent dye or any other applicable cellular marker molecule.Such marker molecules are well known in the art. For example afluorescence-labeling, for example provided by a fluorescence dye, canprovide a visualization of the bound aptamer by fluorescence or laserscanning microscopy or flow cytometry.

Each scaffold can be conjugated with a second active molecule such asfor example IL-21, anti-CD3, anti-CD28.

For further information on polypeptide scaffolds see for example thebackground section of WO 2014/071978A1 and the references cited therein.

The present invention further relates to aptamers. Aptamers (see forexample WO 2014/191359 and the literature as cited therein) are shortsingle-stranded nucleic acid molecules, which can fold into definedthree-dimensional structures and recognize specific target structures.They have appeared to be suitable alternatives for developing targetedtherapies. Aptamers have been shown to selectively bind to a variety ofcomplex targets with high affinity and specificity.

Aptamers recognizing cell surface located molecules have been identifiedwithin the past decade and provide means for developing diagnostic andtherapeutic approaches. Since aptamers have been shown to possess almostno toxicity and immunogenicity they are promising candidates forbiomedical applications. Indeed aptamers, for example prostate-specificmembrane-antigen recognizing aptamers, have been successfully employedfor targeted therapies and shown to be functional in xenograft in vivomodels. Furthermore, aptamers recognizing specific tumor cell lines havebeen identified.

DNA aptamers can be selected to reveal broad-spectrum recognitionproperties for various cancer cells, and particularly those derived fromsolid tumors, while non-tumorigenic and primary healthy cells are notrecognized. If the identified aptamers recognize not only a specifictumor sub-type but rather interact with a series of tumors, this rendersthe aptamers applicable as so-called broad-spectrum diagnostics andtherapeutics.

Further, investigation of cell-binding behavior with flow cytometryshowed that the aptamers revealed very good apparent affinities that arewithin the nanomolar range.

Aptamers are useful for diagnostic and therapeutic purposes. Further, itcould be shown that some of the aptamers are taken up by tumor cells andthus can function as molecular vehicles for the targeted delivery ofanti-cancer agents such as siRNA into tumor cells.

Aptamers can be selected against complex targets such as cells andtissues and complexes of the peptides comprising, preferably consistingof, a sequence according to any of SEQ ID NO 1 to SEQ ID NO 549,according to the invention at hand with the MHC molecule, using thecell-SELEX (Systematic Evolution of Ligands by Exponential enrichment)technique.

The peptides of the present invention can be used to generate anddevelop specific antibodies against MHC/peptide complexes. These can beused for therapy, targeting toxins or radioactive substances to thediseased tissue. Another use of these antibodies can be targetingradionuclides to the diseased tissue for imaging purposes such as PET.This use can help to detect small metastases or to determine the sizeand precise localization of diseased tissues.

Therefore, it is a further aspect of the invention to provide a methodfor producing a recombinant antibody specifically binding to a humanmajor histocompatibility complex (MHC) class I or II being complexedwith a HLA-restricted antigen, the method comprising: immunizing agenetically engineered non-human mammal comprising cells expressing saidhuman major histocompatibility complex (MHC) class I or II with asoluble form of a MHC class I or II molecule being complexed with saidHLA-restricted antigen; isolating mRNA molecules from antibody producingcells of said non-human mammal; producing a phage display librarydisplaying protein molecules encoded by said mRNA molecules; andisolating at least one phage from said phage display library, said atleast one phage displaying said antibody specifically binding to saidhuman major histocompatibility complex (MHC) class I or II beingcomplexed with said HLA-restricted antigen.

It is a further aspect of the invention to provide an antibody thatspecifically binds to a human major histocompatibility complex (MHC)class I or II being complexed with a HLA-restricted antigen, wherein theantibody preferably is a polyclonal antibody, monoclonal antibody,bi-specific antibody and/or a chimeric antibody.

Respective methods for producing such antibodies and single chain classI major histocompatibility complexes, as well as other tools for theproduction of these antibodies are disclosed in WO 03/068201, WO2004/084798, WO 01/72768, WO 03/070752, and in publications (Cohen etal., 2003a; Cohen et al., 2003b; Denkberg et al., 2003), which for thepurposes of the present invention are all explicitly incorporated byreference in their entireties.

Preferably, the antibody is binding with a binding affinity of below 20nanomolar, preferably of below 10 nanomolar, to the complex, which isregarded as “specific” in the context of the present invention.

The present invention relates to a peptide comprising a sequence that isselected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 549, ora variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 549 or a variant thereof thatinduces T cells cross-reacting with said peptide, wherein said peptideis not the underlying full-length polypeptide.

The present invention further relates to a peptide comprising a sequencethat is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:549 or a variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 549, wherein said peptide orvariant has an overall length of between 8 and 100, preferably between 8and 30, and most preferred between 8 and 14 amino acids.

The present invention further relates to the peptides according to theinvention that have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or -II.

The present invention further relates to the peptides according to theinvention wherein the peptide consists or consists essentially of anamino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 549.

The present invention further relates to the peptides according to theinvention, wherein the peptide is (chemically) modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to theinvention, wherein the peptide is part of a fusion protein, inparticular comprising N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii), or wherein the peptide is fusedto (or into) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

Another embodiment of the present invention relates to a non-naturallyoccurring peptide wherein said peptide consists or consists essentiallyof an amino acid sequence according to SEQ ID No: 1 to SEQ ID No: 48 andhas been synthetically produced (e.g. synthesized) as a pharmaceuticallyacceptable salt. Methods to synthetically produce peptides are wellknown in the art. The salts of the peptides according to the presentinvention differ substantially from the peptides in their state(s) invivo, as the peptides as generated in vivo are no salts. The non-naturalsalt form of the peptide mediates the solubility of the peptide, inparticular in the context of pharmaceutical compositions comprising thepeptides, e.g. the peptide vaccines as disclosed herein. A sufficientand at least substantial solubility of the peptide(s) is required inorder to efficiently provide the peptides to the subject to be treated.Preferably, the salts are pharmaceutically acceptable salts of thepeptides. These salts according to the invention include alkaline andearth alkaline salts such as salts of the Hofmeister series comprisingas anions PO₄ ³⁻, SO₄ ²⁻, CH₃COO⁻, Cl⁻, Br, NO₃ ⁻, ClO₄ ⁻, I⁻, SCN⁻ andas cations NH₄ ⁺, Rb⁺, K⁺, Na⁺, Cs⁺, Li⁺, Zn²⁺, Mg²⁺, Ca²⁺, Mn²⁺, Cu²⁺and Ba²⁺. Particularly salts are selected from (NH₄)₃PO₄, (NH₄)₂HPO₄,(NH₄)H₂PO₄, (NH₄)₂SO₄, NH₄CH₃COO, NH₄Cl, NH₄Br, NH₄NO₃, NH₄ClO₄, NH₄I,NH₄SCN, Rb₃PO₄, Rb₂HPO₄, RbH₂PO₄, Rb₂SO₄, Rb₄CH₃COO, Rb₄Cl, Rb₄Br,Rb₄NO₃, Rb₄ClO₄, Rb₄I, Rb₄SCN, K₃PO₄, K₂HPO₄, KH₂PO₄, K₂SO₄, KCH₃COO,KCl, KBr, KNOB, KClO₄, KI, KSCN, Na₃PO₄, Na₂HPO₄, NaH₂PO₄, Na₂SO₄,NaCH₃COO, NaCl, NaBr, NaNO₃, NaClO₄, Nal, NaSCN, ZnCl₂ Cs₃PO₄, Cs₂HPO₄,CsH₂PO₄, Cs₂SO₄, CsCH₃COO, CsCl, CsBr, CsNO₃, CsClO₄, CsI, CsSCN,Li₃PO₄, Li₂HPO₄, LiH₂PO₄, Li₂SO₄, LiCH₃COO, LiCl, LiBr, LiNO₃, LiClO₄,LiI, LiSCN, Cu₂SO₄, Mg₃(PO₄)₂, Mg₂HPO₄, Mg(H₂PO₄)₂, Mg₂SO₄, Mg(CH₃COO)₂,MgCl₂, MgBr₂, Mg(NO₃)₂, Mg(ClO₄)₂, Mgl₂, Mg(SCN)₂, MnCl₂, Ca₃(PO₄),Ca₂HPO₄, Ca(H₂PO₄)₂, CaSO₄, Ca(CH₃COO)₂, CaCl₂), CaBr₂, Ca(NO₃)₂,Ca(ClO₄)₂, CaI₂, Ca(SCN)₂, Ba₃(PO₄)₂, Ba₂HPO₄, Ba(H₂PO₄)₂, BaSO₄,Ba(CH₃COO)₂, BaCl₂, BaBr₂, Ba(NO₃)₂, Ba(ClO₄)₂, BaI₂, and Ba(SCN)₂.Particularly preferred are NH acetate, MgCl₂, KH₂PO₄, Na₂SO₄, KCl, NaCl,and CaCl₂), such as, for example, the chloride or acetate(trifluoroacetate) salts.

Generally, peptides and variants (at least those containing peptidelinkages between amino acid residues) may be synthesized by theFmoc-polyamide mode of solid-phase peptide synthesis as disclosed byLukas et al. (Lukas et al., 1981) and by references as cited therein.Temporary N-amino group protection is afforded by the9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of thishighly base-labile protecting group is done using 20% piperidine in N,N-dimethylformamide. Side-chain functionalities may be protected astheir butyl ethers (in the case of serine threonine and tyrosine), butylesters (in the case of glutamic acid and aspartic acid),butyloxycarbonyl derivative (in the case of lysine and histidine),trityl derivative (in the case of cysteine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). Where glutamine or asparagine are C-terminal residues, use ismade of the 4,4′-dimethoxybenzhydryl group for protection of the sidechain amido functionalities. The solid-phase support is based on apolydimethyl-acrylamide polymer constituted from the three monomersdimethylacrylamide (backbone-monomer), bisacryloylethylene diamine(cross linker) and acryloylsarcosine methyl ester (functionalizingagent). The peptide-to-resin cleavable linked agent used is theacid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All aminoacid derivatives are added as their preformed symmetrical anhydridederivatives with the exception of asparagine and glutamine, which areadded using a reversed N, N-dicyclohexyl-carbodiimide/1hydroxybenzotriazole mediated coupling procedure. All coupling anddeprotection reactions are monitored using ninhydrine, trinitrobenzenesulphonic acid or isotin test procedures. Upon completion of synthesis,peptides are cleaved from the resin support with concomitant removal ofside-chain protecting groups by treatment with 95% trifluoroacetic acidcontaining a 50% scavenger mix. Scavengers commonly used includeethanedithiol, phenol, anisole and water, the exact choice depending onthe constituent amino acids of the peptide being synthesized. Also acombination of solid phase and solution phase methodologies for thesynthesis of peptides is possible (see, for example, (Bruckdorfer etal., 2004), and the references as cited therein).

Trifluoroacetic acid is removed by evaporation in vacuo, with subsequenttrituration with diethyl ether affording the crude peptide. Anyscavengers present are removed by a simple extraction procedure which onlyophilization of the aqueous phase affords the crude peptide free ofscavengers. Reagents for peptide synthesis are generally available frome.g. Calbiochem-Novabiochem (Nottingham, UK).

Purification may be performed by any one, or a combination of,techniques such as re-crystallization, size exclusion chromatography,ion-exchange chromatography, hydrophobic interaction chromatography and(usually) reverse-phase high performance liquid chromatography usinge.g. acetonitril/water gradient separation.

The present invention further relates to a nucleic acid, encoding thepeptides according to the invention, provided that the peptide is notthe complete (full) human protein.

The present invention further relates to the nucleic acid according tothe invention that is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing a nucleic acid according to the present invention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use inmedicine, in particular in the treatment of ovarian cancer.

The present invention further relates to a host cell comprising anucleic acid according to the invention or an expression vectoraccording to the invention.

The present invention further relates to the host cell according to thepresent invention that is an antigen presenting cell, and preferably adendritic cell.

The present invention further relates to a method of producing a peptideaccording to the present invention, said method comprising culturing thehost cell according to the present invention, and isolating the peptidefrom said host cell or its culture medium.

The present invention further relates to the method according to thepresent invention, where-in the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellby contacting a sufficient amount of the antigen with anantigen-presenting cell.

The present invention further relates to the method according to theinvention, wherein the antigen-presenting cell comprises an expressionvector capable of expressing said peptide containing SEQ ID NO: 1 to SEQID NO: 549 or said variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellsselectively recognizes a cell which aberrantly expresses a polypeptidecomprising an amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as according to the present invention.

The present invention further relates to the use of any peptidedescribed, a nucleic acid according to the present invention, anexpression vector according to the present invention, a cell accordingto the present invention, or an activated cytotoxic T lymphocyteaccording to the present invention as a medicament or in the manufactureof a medicament. The present invention further relates to a useaccording to the present invention, wherein the medicament is activeagainst cancer.

The present invention further relates to a use according to theinvention, wherein the medicament is a vaccine. The present inventionfurther relates to a use according to the invention, wherein themedicament is active against cancer.

The present invention further relates to a use according to theinvention, wherein said cancer cells are ovarian cancer cells or othersolid or hematological tumor cells such as pancreatic cancer, braincancer, kidney cancer, colon or rectal cancer, leukemia.

The present invention further relates to particular marker proteins andbiomarkers based on the peptides according to the present invention,herein called “targets” that can be used in the diagnosis and/orprognosis of ovarian cancer. The present invention also relates to theuse of these novel targets for cancer treatment.

The term “antibody” or “antibodies” is used herein in a broad sense andincludes both polyclonal and monoclonal antibodies. In addition tointact or “full” immunoglobulin molecules, also included in the term“antibodies” are fragments (e.g. CDRs, Fv, Fab and Fc fragments) orpolymers of those immunoglobulin molecules and humanized versions ofimmunoglobulin molecules, as long as they exhibit any of the desiredproperties (e.g., specific binding of a ovarian cancer markerpolypeptide, delivery of a toxin to a ovarian cancer cell expressing acancer marker gene at an increased level, and/or inhibiting the activityof a ovarian cancer marker polypeptide) according to the invention.

Whenever possible, the antibodies of the invention may be purchased fromcommercial sources. The antibodies of the invention may also begenerated using well-known methods. The skilled artisan will understandthat either full length ovarian cancer marker polypeptides or fragmentsthereof may be used to generate the antibodies of the invention. Apolypeptide to be used for generating an antibody of the invention maybe partially or fully purified from a natural source, or may be producedusing recombinant DNA techniques.

For example, a cDNA encoding a peptide according to the presentinvention, such as a peptide according to SEQ ID NO: 1 to SEQ ID NO:549, or a variant or fragment thereof, can be expressed in prokaryoticcells (e.g., bacteria) or eukaryotic cells (e.g., yeast, insect, ormammalian cells), after which the recombinant protein can be purifiedand used to generate a monoclonal or polyclonal antibody preparationthat specifically bind the ovarian cancer marker polypeptide used togenerate the antibody according to the invention.

One of skill in the art will realize that the generation of two or moredifferent sets of monoclonal or polyclonal antibodies maximizes thelikelihood of obtaining an antibody with the specificity and affinityrequired for its intended use (e.g., ELISA, immunohistochemistry, invivo imaging, immunotoxin therapy). The antibodies are tested for theirdesired activity by known methods, in accordance with the purpose forwhich the antibodies are to be used (e.g., ELISA, immunohistochemistry,immunotherapy, etc.; for further guidance on the generation and testingof antibodies, see, e.g., Greenfield, 2014 (Greenfield, 2014)). Forexample, the antibodies may be tested in ELISA assays or, Western blots,immunohistochemical staining of formalin-fixed lung cancers or frozentissue sections. After their initial in vitro characterization,antibodies intended for therapeutic or in vivo diagnostic use are testedaccording to known clinical testing methods.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e.; the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The monoclonal antibodies herein specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired antagonistic activity (U.S. Pat. No. 4,816,567, which is herebyincorporated in its entirety).

Monoclonal antibodies of the invention may be prepared using hybridomamethods. In a hybridoma method, a mouse or other appropriate host animalis typically immunized with an immunizing agent to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies).

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 and U.S. Pat. No.4,342,566. Papain digestion of antibodies typically produces twoidentical antigen binding fragments, called Fab fragments, each with asingle antigen binding site, and a residual Fc fragment. Pepsintreatment yields a F(ab′)2 fragment and a pFc′ fragment.

The antibody fragments, whether attached to other sequences or not, canalso include insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the fragment is not significantly altered orimpaired compared to the non-modified antibody or antibody fragment.These modifications can provide for some additional property, such as toremove/add amino acids capable of disulfide bonding, to increase itsbio-longevity, to alter its secretory characteristics, etc. In any case,the antibody fragment must possess a bioactive property, such as bindingactivity, regulation of binding at the binding domain, etc. Functionalor active regions of the antibody may be identified by mutagenesis of aspecific region of the protein, followed by expression and testing ofthe expressed polypeptide. Such methods are readily apparent to askilled practitioner in the art and can include site-specificmutagenesis of the nucleic acid encoding the antibody fragment.

The antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′ or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues, which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source, which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed by substituting rodent CDRs or CDR sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

Transgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production can be employed. For example, ithas been described that the homozygous deletion of the antibody heavychain joining region gene in chimeric and germ-line mutant mice resultsin complete inhibition of endogenous antibody production. Transfer ofthe human germ-line immunoglobulin gene array in such germ-line mutantmice will result in the production of human antibodies upon antigenchallenge. Human antibodies can also be produced in phage displaylibraries.

Antibodies of the invention are preferably administered to a subject ina pharmaceutically acceptable carrier. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include saline, Ringer's solutionand dextrose solution. The pH of the solution is preferably from about 5to about 8, and more preferably from about 7 to about 7.5. Furthercarriers include sustained release preparations such as semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles, e.g., films, liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of antibodybeing administered.

The antibodies can be administered to the subject, patient, or cell byinjection (e.g., intravenous, intraperitoneal, subcutaneous,intramuscular), or by other methods such as infusion that ensure itsdelivery to the bloodstream in an effective form. The antibodies mayalso be administered by intra tumoral or peritumoral routes, to exertlocal as well as systemic therapeutic effects. Local or intravenousinjection is preferred.

Effective dosages and schedules for administering the antibodies may bedetermined empirically, and making such determinations is within theskill in the art. Those skilled in the art will understand that thedosage of antibodies that must be administered will vary depending on,for example, the subject that will receive the antibody, the route ofadministration, the particular type of antibody used and other drugsbeing administered. A typical daily dosage of the antibody used alonemight range from about 1 (μg/kg to up to 100 mg/kg of body weight ormore per day, depending on the factors mentioned above. Followingadministration of an antibody, preferably for treating ovarian cancer,the efficacy of the therapeutic antibody can be assessed in various wayswell known to the skilled practitioner. For instance, the size, number,and/or distribution of lung cancer in a subject receiving treatment maybe monitored using standard tumor imaging techniques. Atherapeutically-administered antibody that arrests tumor growth, resultsin tumor shrinkage, and/or prevents the development of new tumors,compared to the disease course that would occurs in the absence ofantibody administration, is an efficacious antibody for treatment oflung cancer.

It is a further aspect of the invention to provide a method forproducing a soluble T-cell receptor (sTCR) recognizing a specificpeptide-MHC complex. Such soluble T-cell receptors can be generated fromspecific T-cell clones, and their affinity can be increased bymutagenesis targeting the complementarity-determining regions. For thepurpose of T-cell receptor selection, phage display can be used (US2010/0113300, (Liddy et al., 2012)). For the purpose of stabilization ofT-cell receptors during phage display and in case of practical use asdrug, alpha and beta chain can be linked e.g. by non-native disulfidebonds, other covalent bonds (single-chain T-cell receptor), or bydimerization domains (Boulter et al., 2003; Card et al., 2004; Willcoxet al., 1999). The T-cell receptor can be linked to toxins, drugs,cytokines (see, for example, US 2013/0115191), domains recruitingeffector cells such as an anti-CD3 domain, etc., in order to executeparticular functions on target cells. Moreover, it could be expressed inT cells used for adoptive transfer. Further information can be found inWO 2004/033685A1 and WO 2004/074322A1. A combination of sTCRs isdescribed in WO 2012/056407A1. Further methods for the production aredisclosed in WO 2013/057586A1.

In addition, the peptides and/or the TCRs or antibodies or other bindingmolecules of the present invention can be used to verify a pathologist'sdiagnosis of a cancer based on a biopsied sample.

The antibodies or TCRs may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radionucleotide (such as¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P or ³⁵S) so that the tumor can belocalized using immunoscintiography. In one embodiment, antibodies orfragments thereof bind to the extracellular domains of two or moretargets of a protein selected from the group consisting of theabove-mentioned proteins, and the affinity value (Kd) is less than 1×10μM.

Antibodies for diagnostic use may be labeled with probes suitable fordetection by various imaging methods. Methods for detection of probesinclude, but are not limited to, fluorescence, light, confocal andelectron microscopy; magnetic resonance imaging and spectroscopy;fluoroscopy, computed tomography and positron emission tomography.Suitable probes include, but are not limited to, fluorescein, rhodamine,eosin and other fluorophores, radioisotopes, gold, gadolinium and otherlanthanides, paramagnetic iron, fluorine-18 and other positron-emittingradionuclides. Additionally, probes may be bi- or multi-functional andbe detectable by more than one of the methods listed. These antibodiesmay be directly or indirectly labeled with said probes. Attachment ofprobes to the antibodies includes covalent attachment of the probe,incorporation of the probe into the antibody, and the covalentattachment of a chelating compound for binding of probe, amongst otherswell recognized in the art. For immunohistochemistry, the disease tissuesample may be fresh or frozen or may be embedded in paraffin and fixedwith a preservative such as formalin. The fixed or embedded sectioncontains the sample are contacted with a labeled primary antibody andsecondary antibody, wherein the antibody is used to detect theexpression of the proteins in situ.

Another aspect of the present invention includes an in vitro method forproducing activated T cells, the method comprising contacting in vitro Tcells with antigen loaded human MHC molecules expressed on the surfaceof a suitable antigen-presenting cell for a period of time sufficient toactivate the T cell in an antigen specific manner, wherein the antigenis a peptide according to the invention. Preferably a sufficient amountof the antigen is used with an antigen-presenting cell.

Preferably the mammalian cell lacks or has a reduced level or functionof the TAP peptide transporter. Suitable cells that lack the TAP peptidetransporter include T2, RMA-S and Drosophila cells. TAP is thetransporter associated with antigen processing.

The human peptide loading deficient cell line T2 is available from theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852, USA under Catalogue No CRL 1992; the Drosophila cell lineSchneider line 2 is available from the ATCC under Catalogue No CRL19863; the mouse RMA-S cell line is described in Ljunggren et al.(Ljunggren and Karre, 1985).

Preferably, before transfection the host cell expresses substantially noMHC class I molecules. It is also preferred that the stimulator cellexpresses a molecule important for providing a co-stimulatory signal forT-cells such as any of B7.1, B7.2, ICAM-1 and LFA 3. The nucleic acidsequences of numerous MHC class I molecules and of the co-stimulatormolecules are publicly available from the GenBank and EMBL databases.

In case of a MHC class I epitope being used as an antigen, the T cellsare CD8-positive T cells.

If an antigen-presenting cell is transfected to express such an epitope,preferably the cell comprises an expression vector capable of expressinga peptide containing SEQ ID NO: 1 to SEQ ID NO: 549, or a variant aminoacid sequence thereof.

A number of other methods may be used for generating T cells in vitro.For example, autologous tumor-infiltrating lymphocytes can be used inthe generation of CTL. Plebanski et al. (Plebanski et al., 1995) madeuse of autologous peripheral blood lymphocytes (PLBs) in the preparationof T cells. Furthermore, the production of autologous T cells by pulsingdendritic cells with peptide or polypeptide, or via infection withrecombinant virus is possible. Also, B cells can be used in theproduction of autologous T cells. In addition, macrophages pulsed withpeptide or polypeptide, or infected with recombinant virus, may be usedin the preparation of autologous T cells. S. Walter et al. (Walter etal., 2003) describe the in vitro priming of T cells by using artificialantigen presenting cells (aAPCs), which is also a suitable way forgenerating T cells against the peptide of choice. In the presentinvention, aAPCs were generated by the coupling of preformed MHC:peptidecomplexes to the surface of polystyrene particles (microbeads) bybiotin:streptavidin biochemistry. This system permits the exact controlof the MHC density on aAPCs, which allows to selectively elicit high- orlow-avidity antigen-specific T cell responses with high efficiency fromblood samples. Apart from MHC:peptide complexes, aAPCs should carryother proteins with co-stimulatory activity like anti-CD28 antibodiescoupled to their surface. Furthermore such aAPC-based systems oftenrequire the addition of appropriate soluble factors, e.g. cytokines,like interleukin-12.

Allogeneic cells may also be used in the preparation of T cells and amethod is described in detail in WO 97/26328, incorporated herein byreference. For example, in addition to Drosophila cells and T2 cells,other cells may be used to present antigens such as CHO cells,baculovirus-infected insect cells, bacteria, yeast, vaccinia-infectedtarget cells. In addition plant viruses may be used (see, for example,Porta et al. (Porta et al., 1994) which describes the development ofcowpea mosaic virus as a high-yielding system for the presentation offoreign peptides.

The activated T cells that are directed against the peptides of theinvention are useful in therapy. Thus, a further aspect of the inventionprovides activated T cells obtainable by the foregoing methods of theinvention.

Activated T cells, which are produced by the above method, willselectively recognize a cell that aberrantly expresses a polypeptidethat comprises an amino acid sequence of SEQ ID NO: 1 to SEQ ID NO 549.

Preferably, the T cell recognizes the cell by interacting through itsTCR with the HLA/peptide-complex (for example, binding). The T cells areuseful in a method of killing target cells in a patient whose targetcells aberrantly express a polypeptide comprising an amino acid sequenceof the invention wherein the patient is administered an effective numberof the activated T cells. The T cells that are administered to thepatient may be derived from the patient and activated as described above(i.e. they are autologous T cells). Alternatively, the T cells are notfrom the patient but are from another individual. Of course, it ispreferred if the individual is a healthy individual. By “healthyindividual” the inventors mean that the individual is generally in goodhealth, preferably has a competent immune system and, more preferably,is not suffering from any disease that can be readily tested for, anddetected.

In vivo, the target cells for the CD8-positive T cells according to thepresent invention can be cells of the tumor (which sometimes express MHCclass II) and/or stromal cells surrounding the tumor (tumor cells)(which sometimes also express MHC class II; (Dengjel et al., 2006)).

The T cells of the present invention may be used as active ingredientsof a therapeutic composition. Thus, the invention also provides a methodof killing target cells in a patient whose target cells aberrantlyexpress a polypeptide comprising an amino acid sequence of theinvention, the method comprising administering to the patient aneffective number of T cells as defined above.

By “aberrantly expressed” the inventors also mean that the polypeptideis over-expressed compared to normal levels of expression or that thegene is silent in the tissue from which the tumor is derived but in thetumor it is expressed. By “over-expressed” the inventors mean that thepolypeptide is present at a level at least 1.2-fold of that present innormal tissue; preferably at least 2-fold, and more preferably at least5-fold or 10-fold the level present in normal tissue.

T cells may be obtained by methods known in the art, e.g. thosedescribed above.

Protocols for this so-called adoptive transfer of T cells are well knownin the art. Reviews can be found in: Gattioni et al. and Morgan et al.(Gattinoni et al., 2006; Morgan et al., 2006).

Another aspect of the present invention includes the use of the peptidescomplexed with MHC to generate a T-cell receptor whose nucleic acid iscloned and is introduced into a host cell, preferably a T cell. Thisengineered T cell can then be transferred to a patient for therapy ofcancer.

Any molecule of the invention, i.e. the peptide, nucleic acid, antibody,expression vector, cell, activated T cell, T-cell receptor or thenucleic acid encoding it, is useful for the treatment of disorders,characterized by cells escaping an immune response. Therefore anymolecule of the present invention may be used as medicament or in themanufacture of a medicament. The molecule may be used by itself orcombined with other molecule(s) of the invention or (a) knownmolecule(s).

Because the underlying polypeptides of the peptides of the invention asmentioned in the Tables above are highly expressed in ovarian cancer,and are expressed at rather to extremely low levels in normal cells,targeting peptides derived from the protein products of the followinggenes may preferably be integrated into a therapeutic strategy:

The present invention further provides a medicament that is useful intreating cancer, in particular ovarian cancer and other malignancies.

The present invention is further directed at a kit comprising:

(a) a container containing a pharmaceutical composition as describedabove, in solution or in lyophilized form;

(b) optionally a second container containing a diluent or reconstitutingsolution for the lyophilized formulation; and

(c) optionally, instructions for (i) use of the solution or (ii)reconstitution and/or use of the lyophilized formulation.

The kit may further comprise one or more of (iii) a buffer, (iv) adiluent, (v) a filter, (vi) a needle, or (v) a syringe. The container ispreferably a bottle, a vial, a syringe or test tube; and it may be amulti-use container. The pharmaceutical composition is preferablylyophilized.

Kits of the present invention preferably comprise a lyophilizedformulation of the present invention in a suitable container andinstructions for its reconstitution and/or use. Suitable containersinclude, for example, bottles, vials (e.g. dual chamber vials), syringes(such as dual chamber syringes) and test tubes. The container may beformed from a variety of materials such as glass or plastic. Preferablythe kit and/or container contain/s instructions on or associated withthe container that indicates directions for reconstitution and/or use.For example, the label may indicate that the lyophilized formulation isto be reconstituted to peptide concentrations as described above. Thelabel may further indicate that the formulation is useful or intendedfor subcutaneous administration.

The container holding the formulation may be a multi-use vial, whichallows for repeat administrations (e.g., from 2-6 administrations) ofthe reconstituted formulation. The kit may further comprise a secondcontainer comprising a suitable diluent (e.g., sodium bicarbonatesolution).

Upon mixing of the diluent and the lyophilized formulation, the finalpeptide concentration in the reconstituted formulation is preferably atleast 0.15 mg/mL/peptide (=75 μg) and preferably not more than 3mg/mL/peptide (=1500 μg). The kit may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use.

Kits of the present invention may have a single container that containsthe formulation of the pharmaceutical compositions according to thepresent invention with or without other components (e.g., othercompounds or pharmaceutical compositions of these other compounds) ormay have distinct container for each component.

Preferably, kits of the invention include a formulation of the inventionpackaged for use in combination with the co-administration of a secondcompound (such as adjuvants (e.g. GM-CSF), a chemotherapeutic agent, anatural product, a hormone or antagonist, an anti-angiogenesis agent orinhibitor, an apoptosis-inducing agent or a chelator) or apharmaceutical composition thereof. The components of the kit may bepre-complexed or each component may be in a separate distinct containerprior to administration to a patient. The components of the kit may beprovided in one or more liquid solutions, preferably, an aqueoussolution, more preferably, a sterile aqueous solution. The components ofthe kit may also be provided as solids, which may be converted intoliquids by addition of suitable solvents, which are preferably providedin another distinct container.

The container of a therapeutic kit may be a vial, test tube, flask,bottle, syringe, or any other means of enclosing a solid or liquid.Usually, when there is more than one component, the kit will contain asecond vial or other container, which allows for separate dosing. Thekit may also contain another container for a pharmaceutically acceptableliquid. Preferably, a therapeutic kit will contain an apparatus (e.g.,one or more needles, syringes, eye droppers, pipette, etc.), whichenables administration of the agents of the invention that arecomponents of the present kit.

The present formulation is one that is suitable for administration ofthe peptides by any acceptable route such as oral (enteral), nasal,ophthal, subcutaneous, intradermal, intramuscular, intravenous ortransdermal. Preferably, the administration is s.c., and most preferablyi.d. administration may be by infusion pump.

Since the peptides of the invention were isolated from ovarian cancer,the medicament of the invention is preferably used to treat ovariancancer.

The present invention further includes a method for producing apersonalized pharmaceutical for an individual patient comprisingmanufacturing a pharmaceutical composition comprising at least onepeptide selected from a warehouse of pre-screened TUMAPs, wherein the atleast one peptide used in the pharmaceutical composition is selected forsuitability in the individual patient. In one embodiment, thepharmaceutical composition is a vaccine. The method could also beadapted to produce T cell clones for down-stream applications, such asTCR isolations, or soluble antibodies, and other treatment options.

A “personalized pharmaceutical” shall mean specifically tailoredtherapies for one individual patient that will only be used for therapyin such individual patient, including actively personalized cancervaccines and adoptive cellular therapies using autologous patienttissue.

As used herein, the term “warehouse” shall refer to a group or set ofpeptides that have been pre-screened for immunogenicity and/orover-presentation in a particular tumor type. The term “warehouse” isnot intended to imply that the particular peptides included in thevaccine have been pre-manufactured and stored in a physical facility,although that possibility is contemplated. It is expressly contemplatedthat the peptides may be manufactured de novo for each individualizedvaccine produced, or may be pre-manufactured and stored. The warehouse(e.g. in the form of a database) is composed of tumor-associatedpeptides, which were highly overexpressed in the tumor tissue of ovariancancer patients with various HLA-A HLA-B and HLA-C alleles. It maycontain MHC class I and MHC class II peptides or elongated MHC class Ipeptides. In addition to the tumor associated peptides collected fromseveral ovarian cancer tissues, the warehouse may contain HLA-A*02 andHLA-A*24 as well as HLAs with smaller abundance marker peptides. Thesepeptides allow comparison of the magnitude of T-cell immunity induced byTUMAPS in a quantitative manner and hence allow important conclusion tobe drawn on the capacity of the vaccine to elicit anti-tumor responses.Secondly, they function as important positive control peptides derivedfrom a “non-self” antigen in the case that any vaccine-induced T-cellresponses to TUMAPs derived from “self” antigens in a patient are notobserved. And thirdly, it may allow conclusions to be drawn, regardingthe status of immuno-competence of the patient.

TUMAPs for the warehouse are identified by using an integratedfunctional genomics approach combining gene expression analysis, massspectrometry, and T-cell immunology (XPresident®). The approach assuresthat only TUMAPs truly present on a high percentage of tumors but not oronly minimally expressed on normal tissue, are chosen for furtheranalysis. For initial peptide selection, ovarian cancer samples frompatients and blood from healthy donors were analyzed in a stepwiseapproach:

1. HLA ligands from the malignant material were identified by massspectrometry

2. Genome-wide messenger ribonucleic acid (mRNA) expression analysis wasused to identify genes over-expressed in the malignant tissue (ovariancancer) compared with a range of normal organs and tissues

3. Identified HLA ligands were compared to gene expression data.Peptides over-presented or selectively presented on tumor tissue,preferably encoded by selectively expressed or over-expressed genes asdetected in step 2 were considered suitable TUMAP candidates for amulti-peptide vaccine.4. Literature research was performed in order to identify additionalevidence supporting the relevance of the identified peptides as TUMAPs5. The relevance of over-expression at the mRNA level was confirmed byredetection of selected TUMAPs from step 3 on tumor tissue and lack of(or infrequent) detection on healthy tissues.6. In order to assess, whether an induction of in vivo T-cell responsesby the selected peptides may be feasible, in vitro immunogenicity assayswere performed using human T cells from healthy donors as well as fromovarian cancer patients.

In an aspect, the peptides are pre-screened for immunogenicity beforebeing included in the warehouse. By way of example, and not limitation,the immunogenicity of the peptides included in the warehouse isdetermined by a method comprising in vitro T-cell priming throughrepeated stimulations of CD8+ T cells from healthy donors withartificial antigen presenting cells loaded with peptide/MHC complexesand anti-CD28 antibody.

This method is preferred for rare cancers and patients with a rareexpression profile. In contrast to multi-peptide cocktails with a fixedcomposition as currently developed, the warehouse allows a significantlyhigher matching of the actual expression of antigens in the tumor withthe vaccine. Selected single or combinations of several “off-the-shelf”peptides will be used for each patient in a multitarget approach. Intheory an approach based on selection of e.g. 5 different antigenicpeptides from a library of 50 would already lead to approximately 17million possible drug product (DP) compositions.

In an aspect, the peptides are selected for inclusion in the vaccinebased on their suitability for the individual patient based on themethod according to the present invention as described herein, or asbelow.

The HLA phenotype, transcriptomic and peptidomic data is gathered fromthe patient's tumor material, and blood samples to identify the mostsuitable peptides for each patient containing “warehouse” andpatient-unique (i.e. mutated) TUMAPs. Those peptides will be chosen,which are selectively or over-expressed in the patients tumor and, wherepossible, show strong in vitro immunogenicity if tested with thepatients' individual PBMCs.

Preferably, the peptides included in the vaccine are identified by amethod comprising: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; (b) comparingthe peptides identified in (a) with a warehouse (database) of peptidesas described above; and (c) selecting at least one peptide from thewarehouse (database) that correlates with a tumor-associated peptideidentified in the patient. For example, the TUMAPs presented by thetumor sample are identified by: (a1) comparing expression data from thetumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. Preferably, the sequences of MHCligands are identified by eluting bound peptides from MHC moleculesisolated from the tumor sample, and sequencing the eluted ligands.Preferably, the tumor sample and the normal tissue are obtained from thesame patient.

In addition to, or as an alternative to, selecting peptides using awarehousing (database) model, TUMAPs may be identified in the patient denovo, and then included in the vaccine. As one example, candidate TUMAPsmay be identified in the patient by (a1) comparing expression data fromthe tumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. As another example, proteins may beidentified containing mutations that are unique to the tumor samplerelative to normal corresponding tissue from the individual patient, andTUMAPs can be identified that specifically target the mutation. Forexample, the genome of the tumor and of corresponding normal tissue canbe sequenced by whole genome sequencing: For discovery of non-synonymousmutations in the protein-coding regions of genes, genomic DNA and RNAare extracted from tumor tissues and normal non-mutated genomic germlineDNA is extracted from peripheral blood mononuclear cells (PBMCs). Theapplied NGS approach is confined to the re-sequencing of protein codingregions (exome re-sequencing). For this purpose, exonic DNA from humansamples is captured using vendor-supplied target enrichment kits,followed by sequencing with e.g. a HiSeq2000 (Illumina). Additionally,tumor mRNA is sequenced for direct quantification of gene expression andvalidation that mutated genes are expressed in the patients' tumors. Theresultant millions of sequence reads are processed through softwarealgorithms. The output list contains mutations and gene expression.Tumor-specific somatic mutations are determined by comparison with thePBMC-derived germline variations and prioritized. The de novo identifiedpeptides can then be tested for immunogenicity as described above forthe warehouse, and candidate TUMAPs possessing suitable immunogenicityare selected for inclusion in the vaccine.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient by the method asdescribed above; (b) comparing the peptides identified in a) with awarehouse of peptides that have been prescreened for immunogenicity andoverpresentation in tumors as compared to corresponding normal tissue;(c) selecting at least one peptide from the warehouse that correlateswith a tumor-associated peptide identified in the patient; and (d)optionally, selecting at least one peptide identified de novo in (a)confirming its immunogenicity.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; and (b)selecting at least one peptide identified de novo in (a) and confirmingits immunogenicity.

Once the peptides for a personalized peptide based vaccine are selected,the vaccine is produced. The vaccine preferably is a liquid formulationconsisting of the individual peptides dissolved in between 20-40% DMSO,preferably about 30-35% DMSO, such as about 33% DMSO.

Each peptide to be included into a product is dissolved in DMSO. Theconcentration of the single peptide solutions has to be chosen dependingon the number of peptides to be included into the product. The singlepeptide-DMSO solutions are mixed in equal parts to achieve a solutioncontaining all peptides to be included in the product with aconcentration of ˜2.5 mg/ml per peptide. The mixed solution is thendiluted 1:3 with water for injection to achieve a concentration of 0.826mg/ml per peptide in 33% DMSO. The diluted solution is filtered througha 0.22 μm sterile filter. The final bulk solution is obtained.

Final bulk solution is filled into vials and stored at −20° C. untiluse. One vial contains 700 μL solution, containing 0.578 mg of eachpeptide. Of this, 500 μL (approx. 400 μg per peptide) will be appliedfor intradermal injection.

In addition to being useful for treating cancer, the peptides of thepresent invention are also useful as diagnostics. Since the peptideswere generated from ovarian cancer cells and since it was determinedthat these peptides are not or at lower levels present in normaltissues, these peptides can be used to diagnose the presence of acancer.

The presence of claimed peptides on tissue biopsies in blood samples canassist a pathologist in diagnosis of cancer. Detection of certainpeptides by means of antibodies, mass spectrometry or other methodsknown in the art can tell the pathologist that the tissue sample ismalignant or inflamed or generally diseased, or can be used as abiomarker for ovarian cancer. Presence of groups of peptides can enableclassification or sub-classification of diseased tissues.

The detection of peptides on diseased tissue specimen can enable thedecision about the benefit of therapies involving the immune system,especially if T-lymphocytes are known or expected to be involved in themechanism of action. Loss of MHC expression is a well describedmechanism by which infected of malignant cells escapeimmuno-surveillance. Thus, presence of peptides shows that thismechanism is not exploited by the analyzed cells.

The peptides of the present invention might be used to analyzelymphocyte responses against those peptides such as T cell responses orantibody responses against the peptide or the peptide complexed to MHCmolecules. These lymphocyte responses can be used as prognostic markersfor decision on further therapy steps. These responses can also be usedas surrogate response markers in immunotherapy approaches aiming toinduce lymphocyte responses by different means, e.g. vaccination ofprotein, nucleic acids, autologous materials, adoptive transfer oflymphocytes. In gene therapy settings, lymphocyte responses againstpeptides can be considered in the assessment of side effects. Monitoringof lymphocyte responses might also be a valuable tool for follow-upexaminations of transplantation therapies, e.g. for the detection ofgraft versus host and host versus graft diseases.

The present invention will now be described in the following examples,which describe preferred embodiments thereof, nevertheless, withoutbeing limited thereto. For the purposes of the present invention, allreferences as cited herein are incorporated by reference in theirentireties.

FIGURES

FIGS. 1A and 1B show the HLA-A,B,C (FIG. 1A) and HLA-DR (FIG. 1B)expression of different cell subsets within ovarian cancer and benignovarian tissue. For FIG. 1 the two-tailed unpaired Student's t-test withWelch's correction was used owing to unequal variance between the twocomparison groups. HLA class I (FIG. 1A) and HLA-DR (FIG. 1B) expressionon different cell types within EOC and benign ovarian tissue afterenzymatic dissociation characterized by distinct cell surface markers(leukocyte compartments: CD45+, tumor cells/epithelial cellcompartments: CD45-EpCam+, endothelial cell compartments: CD45-CD31+).Each data point represents the mean of triplicate experiments performedfor each sample. Two sided t-tests were used to test for significance(*p<0.05; **p<0.01).

FIGS. 2A to 2D show the comparative profiling of the immunopeptidome ofEOC vs. benign tissues. (FIG. 2A) Comparative profiling of HLA class Iligand source proteins represented in EOC (n=34) and benign tissues. Thefrequency of HLA restricted presentation of source proteins is indicatedon the y-axis separately for EOC (above x-axis) and benign sources(below x-axis). The source proteins were ranked (from left to right)according to their frequency of EOC specific presentation. The box onthe left side highlights the TOP100 HLA ligand source proteinsexclusively presented by EOC. (FIG. 2B) Word cloud of the TOP 100 EOCspecific HLA class I ligand source proteins (uniprot recommended genename). Font size (5-26) correlates with absolute number of cancerpatients presenting HLA ligands of respective source proteins. (FIG. 2C)Comparative profiling of HLA class II ligand source proteins representedin EOC (n=22) and benign tissues. (FIG. 2D) Word cloud of the TOP 100EOC specific HLA class II ligand source proteins (uniprot recommendedgene name). Font size (3-11) correlates with absolute number of cancerpatients presenting HLA ligands of respective source proteins.

FIGS. 3A and 3B show the cellular origin of the TOP100 EOC associatedHLA class I ligands. Volcano plots of the relative abundance of HLAligands in the class I immunopeptidome of enriched cell populations ofOvCa 84 analyzed by label free quantitation. Panels show on the leftside (FIG. 3A) tumor infiltrating leukocytes (CD45+) vs. tumor cells(CD45-Epcam+) and on the right side (FIG. 3B) stroma cells (CD45-EpCam−)vs. tumor cells. The horizontal dashed line indicates significancethreshold (p<0.05). TOP100 EOC exclusive ligands (MUC16 (red), DDR1,EYA2, SOX9, TLR7, OASL) as well as ligands derived from leukocyteassociated antigens (CD132, CD8, LSP1) and stroma (endothelial cell)associated antigens (vWF) are highlighted.

FIGS. 4A-4D show the immunohistochemical staining and serum levels assurrogate markers for ligand presentation. Immunohistochemical stainingof high-grade serous ovarian carcinomas for MUC16 (CA-125) with low(IRS4), intermediate (IRS6) and high (IRS12) immunoreactivity score(FIG. 4A). Immunohistochemical staining for Mesothelin (right, IRS8) andIDO1 (left, IRS 12; all at 200× magnification) (FIG. 4B). Correlation ofHLA ligand presentation and source protein expression of selected TOP100EOC associated antigens. Expression of MUC16 (n=23), IDO1 (n=23) andMSLN (n=16) was analyzed by immunohistochemical staining (FIG. 4C) orserum marker analysis of CA-125 (n=30) at the day of surgery (FIG. 4D).For MSLN only the cases for which HLA class II immunopeptidome data wereavailable were included. Non parametric Mann-Whitney test was employedto test for statistical significance (p<0.05 was consideredsignificant).

FIGS. 5A-5C show the prognostic relevance of MUC16 and MSLN.

Immunohistochemical stainings were performed on TMAs with 71 high-gradeserous EOC samples from patients with documented optimal tumordebulking. (FIG. 5A) Kaplan Meier plot depicting the influence of MUC16expression (left panel, low expression score <7, n=41; high expressionscore ≥7, n=30) and MSLN expression (right panel, low expression <6,n=15; high expression ≥6, n=52) on overall survival. (FIG. 5B) Impact ofCD3 T-cell infiltration into the intraepithelial compartment (left panelCD3E, low infiltration <7 cell/HPF, n=13; high infiltration ≥7, n=57) orthe fibrovascular stroma (right panel, CD3S, low infiltration <7cell/HPF, n=40; high infiltration ≥7, n=30) on overall survival ofpatients. (FIG. 5C) Subgroup analysis of combined CD3 and MLN staining(all scoring cutoffs as above) for intraepithelial CD3 T-cells (toppanel, low MSLN/high CD3E, n=11; low MSLN/low CD3E, n=40; high MSLN/lowCD3E, n=14; high MSLN/high CD3E, n=1) or fibrovascular CD3 T-cells(bottom panel, low MSLN/high CD3S, n=30; high MSLN/low CD3S, n=7; lowMSLN/low CD3S, n=21; high MSLN/high CD3S, n=8).

FIG. 6 shows the flow cytometric analysis of EOC and benign ovariantissue. Exemplary presentation of the gating strategy for OvCa 48showing the selection of CD45+ leukocytes, CD45-CD31+ endothelial cellsand CD45-EpCam+ tumor or epithelial cells.

FIGS. 7A and 7B show the saturation analysis of HLA ligand sourceprotein identifications for EOC. Saturation analysis for identificationsof source proteins is depicted separately for HLA class I (FIG. 7A) andHLA class II (FIG. 7B) ligand proteins. The mean number of unique sourceproteins has been calculated for each source count by 1000 randomsamplings from the 34 EOC sources. Exponential regression was used todetermine the calculated maximal attainable coverage of source proteinaccession (dotted lines) for EOC.

FIG. 8 shows the frequency and number of HLA ligand presentation amongEOC samples. HLA presentation of selected EOC associated antigens aswell as the number of different HLA presented peptides (color coding) isvisualized for each individual EOC (patient number on top of eachcolumn) both for class I (top) and class II (bottom) antigens.

EXAMPLES

Materials and Methods

Tissue Samples

All tissue samples were collected at the University Hospital of Tübingenafter obtaining patient informed consent in accordance with theprinciples of the Declaration of Helsinki. All study protocols wereapproved by the local institutional review board. If not statedotherwise samples were stored at −80° C. until further usage. Two-digitHLA typing was performed by sequence specific primer (SSP) PCR using theHLA-Ready Gene System (Innotrain, Kronberg, Germany) and evaluated bySCORE Software (Olerup, Stockholm, Sweden) at the Department ofTransfusion Medicine of the University Hospital of Tübingen. Highresolution four-digit HLA typing was performed by next generationsequencing on a GS Junior Sequencer using the GS GType HLA Primer Sets(both Roche, Basel, Switzerland). Normal tissues were obtained fromBio-Options Inc, CA, USA; BioServe, Beltsville, Md., USA; CapitalBioScience Inc, Rockville, Md., USA; Geneticist Inc., Glendale, Calif.,USA; University Hospital of Geneva; University Hospital of Heidelberg;University Hospital Munich; ProteoGenex Inc., Culver City, Calif., USA;University Hospital of Tübingen. Written informed consents of allpatients had been given before surgery or autopsy. Tissues wereshock-frozen immediately after excision and stored until isolation ofTUMAPs at −70° C. or below.

Tissue Dissociation

EOC as well as benign ovary and fallopian tube tissues were freshlycollected from patients undergoing tumor resection/debulking orsalpingoophorectomy. Tissues were minced into small pieces <2 mm³ andtransferred into an enzymatic dissociation solution containing 400 U/mlCollagenase Type IV, 5 U/ml Dispase (both life technologies, Carlsbad,Calif.) and 0.1 mg/ml DNAse (Roche, Basel, Switzerland) in DMEM (lifetechnologies) with 10% fetal calf serum (Lonza, Basel, Switzerland).Dissociation was performed on a rotating shaker (Infors HT, Basel,Switzerland) for 3 hours at 37° C. Remaining tissue fragments (typically<1% of initial weight) were removed using a 100 μm cell strainer (BD,Franklin Lakes, N.J.). Single cell suspensions were washed twice withPBS and erythrocytes were lysed using ammonium chloride lysis buffer.

HLA Surface Molecule Quantification

HLA surface expression was determined using QIFIKIT quantification flowcytometric assay (Dako, Glostrup, Denmark) according to manufacturer'sinstructions. Cells were stained with either pan-HLA class I specificmonoclonal antibody W6/32, HLA-DR specific L243 or respective isotypecontrol. Discrimination of cell types was based on surface markerstaining with fluorescently labeled antibodies directed against CD45(AmCyan clone 2D1, BD), CD31 (PeCy7, clone WM59, Biolegend, San Diego,Calif.), EpCam (APC, clone HEA125, Miltenyi, Bergisch-Gladbach, Germany)and CD34 (APCCy7,clone 581, Biolegend). 7-AAD (BioLegend) was added asviability marker immediately before analysis on a LSR SORP Fortessainstrument (BD). Triplicates were recorded for each sample with medianfluorescence intensities used for calculation of surface moleculeexpression.

Cell Separation:

Cell separation was performed using two consecutive magnetic activatedcell separation (MACS) protocols according to manufacturer'sinstructions (Miltenyi). Separations were performed using XS columns anda superMACS separator (both Miltenyi). The first separation aimed atpositive selection of CD45⁺ leukocytes. The negative fraction wassubsequently enriched for EpCam⁺ tumor cells. The remaining CD45⁻ EpCam⁻fraction was assumed to represent the stroma cell fraction.

HLA Ligand Isolation

HLA class I and II molecules were isolated by standard immunoaffinitypurification as described previously⁴². Pan-HLA class I specific mAbW6/32 was employed for HLA class I isolation and pan-HLA class II mAbTü39 as well as HLA-DR specific mAb L243 were used for HLA class IIisolation.

Immunopeptidome Analysis by LC-MS/MS

Immunopeptidome analysis was performed on an LTQ OrbitrapXL massspectrometer (Thermo Fisher, Waltham, Mass.) equipped with ananoelectron spray ion source and coupled to an Ultimate 3000 RSLC NanoUHPLC System (Dionex, Sunnyvale, Calif.). Peptide samples were loadedwith 3% of solvent B (20% H₂O, 80% acetonitrile and 0.04% formic acid)on a 2 cm PepMap 100 C18 Nanotrap column (Dionex) at a flow rate of 4μL/min for 10 min. Separation was performed on a 50 cm PepMap C18 columnwith a particle size of 2 μm (Dionex) mounted in a column oven runningat 50° C. The applied gradient ranged from 3 to 30% solvent B within 140min at a flow rate of 175 nil/min. (Solvent A: 99% H₂O, 1% ACN and 0.1%formic acid; Solvent B: 20% H2O, 80% ACN and 0.1% formic acid). Massspectrometry analysis was performed in data dependent acquisition modeemploying a top five method (i.e. during each survey scan the five mostabundant precursor ions were selected for fragmentation). Survey scanswere recorded in the Orbitrap at a resolution of 60,000. MS/MS analysiswas performed by collision induced dissociation (CID, normalizedcollision energy 35%, activation time 30 ms, isolation width 1.3 m/z)with subsequent analysis in the linear trap quadrupole (LTQ). Mass rangefor HLA class I ligands was limited to 400-650 m/z with possible chargestates 2+ and 3+ selected for fragmentation. For HLA class II mass rangewas set to 300-1500 m/z allowing for fragmentation with positive chargestates 2.

HLA class I samples were analyzed in 5 technical replicates while forHLA class II samples 3 technical replicates were typically acquired.Initial runs were performed without dynamic exclusion, whereas forconsecutive runs a dynamic exclusion of 5s was enabled.

Mass Spectrometry Data Processing and Analysis

MS data analysis was carried out using Proteome discoverer 1.3(ThermoFisher). Peak lists were searched against the human proteome ascomprised in the Swiss-Prot database (released Sep. 27, 2013; including20,279 reviewed protein sequences) using Mascot search engine (Mascot2.2.04, Matrix Science, Boston, Mass.). Mass tolerance for processingwas 5 ppm for precursor ions and 0.5 Da for fragment ions. No cleavagespecificity was selected and the only dynamic modification allowed wasoxidized methionine. Peptide confidence was determined using percolatoralgorithm with a target value of q≤0.05 (5% FDR). Additional postprocessing filters were a Mascot Ionscore≥20, search engine rank=1 andpeptide length of 8-12 amino acids for HLA class I ligands and 12-25amino acids for HLA class II ligands. Protein grouping was disabled toensure multiple annotations of peptides, if sequences map into multipleproteins due to conservation. HLA annotation was performed using HLAprediction algorithms hosted at SYFPEITHI and NETMHC 3.4. In case ofambiguous results multiple alleles are mentioned. For comparativeprofiling “one hit wonders” i.e. peptides only presented on one sourcewith a PSM count 5 were removed from both of the datasets.

Label free quantitation of peptides on tumor vs. CD45⁺ and tumor vs.stroma cells was performed using Sieve 2.1 (Thermo Fisher). At least 3replicates of MS raw files for each cell enriched fraction as well asresults from whole tissue MHC precipitations were aligned altogetherwith a maximum retention time (RT) shift of 2.5 mins. Frames weregenerated based on MS² scan events with a maximum RT width of 3.5 minsand 5 ppm mass tolerance. Identifications were imported from Proteomediscoverer using Mascot search results (see above). Total ion currentchromatogram normalization was used to accommodate for differences insample intensities.

Immunogenicity Analysis of HLA Class I Ligands

Priming of peptide specific cytotoxic lymphocytes (CTLs) was conductedusing an established protocol involving artificial antigen presentingcells (aAPCs) (30). aAPCs consisted of streptavidin-coated polystyrenebeads (5.6 μm in diameter; Bangs Laboratories, Fishers, Ind.). Beadswere resuspended at 2×10⁶ particles per ml and incubated with 10 nMbiotinylated peptide-MHC complexes and 10 nM stimulating anti-CD28antibody (clone 9.3 derived from ATCC, Manassas, Va.) each for 30 min atambient temperature. T cells were isolated from whole blood of healthydonors using a CD8 magnetic cell isolation kit (Miltenyi). One millionT-cells per well were cultured in 96 well plates (Corning, Corning,N.Y., USA) and stimulated with the same number of loaded aAPCs in thepresence of 5 ng/ml IL-12 (PromoCell, Heidelberg, Germany). T cells werestimulated 3 times in total with weekly stimulation interval. 40 U/mlIL-2 was added 2 days subsequent to each stimulation. T-cell priming wasassessed by MHC-multimer staining one week after the last stimulationround.

Construction of Tissue Microarrays (TMA)

Consecutive paraffin embedded tumor samples of patients with high-gradeserous carcinoma of the ovary or fallopian tube (EOC) with at least FIGOstage II-III and operated at the University Women's Hospital in Tübingenbetween 1999 and 2008 were retrieved from the archives of the Instituteof Pathology. After confirmation of histological subtype and gradingaccording to published criteria (43). 154 cases were initially includedin the study. A tissue microarray (TMA) was constructed as describedpreviously (44). We used six cores of 0.6 mm diameter of each patient(maximum three cores each from two different sites of the primarytumors—at least two separate cores). In addition we constructed a TMAusing paraffin embedded tissue from the primary tumors of theprospectively collected cases for ligandome analysis. 3 μm thicksections were cut, rehydrated and subjected to specific pretreatment forimmunohistochemistry. In total 23 cases were evaluable for immunoscoringand correlation with immunopeptidome data.

Immunohistochemistry

The following primary antibodies and dilutions were used forimmunohistochemistry: CD3 (1:100, rat monoclonal SP7, DCS, Hamburg,Germany), CD8 (1:200, mouse monoclonal C8/144B, DAKO), MUC16 (1:450,mouse monoclonal M11, DAKO, Glostrup, Denmark), IDO1 (1:25, mousemonoclonal, ABCAM, Cambridge, UK) and MSLN (1:100, mouse monoclonalSPM143, GeneTex, Irvine, Calif., USA). The tissue sections werepre-treated with EDTA-buffer solution (pH 8.6) at 95° C. for 36 min.Immunohistochemical staining was performed on an automated immunostaineraccording to the manufacturer's instructions using the iView DABdetection kit (both Ventana, Tucson, Ariz., USA).

Immunoscoring

Quantification of TILs was carried out by first assessing the averagenumber of immunostained cells per high power field (HPF=400×) bycounting at least 2 HPF for each core. In a second step, the averagenumber of lymphocytes per HPF for the left and right triple core set wascalculated, and for all cores together. This bilateral average count wasused for further calculations. The fibro vascular tumor stroma (CD3S andCD8S), and the intraepithelial compartment of the tumor (CD3E and CD8E)were evaluated separately.

For expression of CA 125, IDO1 and MSLN staining intensity was gradedfrom 0-3, multiplied by a score from 1-4 for the percentage of tumorcells (1: 0-10%; 2: 10-50%; 3: 50-80%; 4: 80-100%). For all parametersthe cases were separated in quartiles and the best separation betweentwo quartiles defined as cut-off value between high and low expression.Of the 154 cases on the TMA 71 patients had undergone documented optimaltumor debulking (<1 cm residual tumor mass) and could be successfullyevaluated for TILs and expression of proteins. Immunoscoring andclinical data analysis were performed by independent investigators.

Statistical Analysis/Visualization

If not mentioned otherwise all figures and statistical analyses weregenerated using Graphpad Prism 6.0 (Graphpad software, La Jolla, Calif.,USA) or Microsoft Office 2010 (Microsoft). Word clouds were createdusing an online applet. Kaplan-Meier analysis was performed using SPSSstatistical software (Version 21, IBM Corp., Armonk, N.Y., USA).Two-tailed unpaired student's t-test was performed unless otherwisespecified. P values less than 0.05 were considered statisticallysignificant. D'Agostino-Pearson omnibus test was used to verifynormality and the F-Test was used to verify equal variance. For FIGS. 1Aand 1B the two-tailed unpaired Student's t-test with Welch's correctionwas used owing to unequal variance between the two comparison groups.Non-parametric Mann-Whitney-test was used in FIGS. 4A-4D because normaldistribution could not be assessed in all cases due to small samplesizes. Spearman correlation was used to correlate IHC scores of MSLN andMUC16 as the datasets were not showing normal distribution. P valuescomparing two Kaplan-Meier survival curves in FIGS. 5A-5C werecalculated using the log-rank (Mantel-Cox) test in Graphpad Prism.

Example 1: HLA Count on Cell Surface and HLA Typing

A major prerequisite for the development of T-cell mediatedimmunotherapies is the expression of MHC molecules on the surface oftumor cells. Therefore, the inventors analyzed and quantified the numberof HLA-A, B, C as well as HLA-DR molecules by flow cytometry ondifferent cell subsets of ovarian tumors (n=11) as well as benigntissues from ovary and fallopian tube (n=8) obtained by enzymaticdissociation. The analysis aimed at the separate quantification of celltype specific HLA expression for leukocytes (CD45^(±)), tumor/epithelialcells (Epcam⁺), and endothelial cells (CD31⁺; the latter only in asubset of 7 ovarian tumors). For the complete gating strategy see FIG.6. The median number of HLA molecules per cell was heterogeneous bothamong different cell types and individual patients, ranging from ˜5,000to 150,000 HLA class I and ˜500 to 330,000 HLA-DR molecules. The numberof HLA-A, B, and C molecules was significantly higher (p=0.0205) onleukocytes isolated from tumor vs. benign tissue indicating an ongoinginflammatory reaction within the tumor. Strong differences in HLA classI expression were also seen when comparing tumor cells with epithelialcells derived from benign tissues. HLA class I molecule expression wassignificantly (p=0.0021) higher on tumor cells (˜75,000 molecules/cell)but remained in the range of other stromal cells such as endothelialcells (˜95,000 molecules/cell). Surprisingly the inventors evidenced astrong (˜105,000 molecules/cell) to some extent extraordinarily highexpression of HLA-DR on EOC cells (>300,000 molecules/cell), whereasbenign epithelial cells were virtually negative for HLA-DR (p=0.0108).Altogether, the inventors could observe an increased MHC class I andclass II expression within the tumors.

HLA ligandome analysis and comparative profiling reveal EOC specificantigen presentation. In order to map the HLA ligand repertoire of EOCthe inventors isolated HLA molecules from bulk tumor tissue andperformed mass spectrometry to characterize the HLA ligandome for atotal of 34 EOCs (for patient characteristics and HLA typing see Table7).

TABLE 7 OvCa Tumor TNM HLA typing ID Age Type Staging MHC class I HLAtyping MHC class II OvCa 9 65 serous T3cNxM1G2R1 A*02:01, DQB1*03:01,DQA1*03:01, ovarian A*03:01, DQA1*05:01, DRB1*11:01, carcinoma B*07:02,DRB1*04:01, DRB3*02:02, B*40:02, DRB4*01:01, DPB1′02:01, C*07:02,DPB1*13:01 C*12:01 OvCa 60 serous T3bN1M1G2R1 A*02:01, DQB1*02:02,DQB1*05:01, 10 ovarian A*11:01, DQA1*01:01, DQA1*03:01, carcinomaB*44:05, DRB1*01:01, DRB1*09:01, B*51:01, DRB4*01:01, DPB1*04:01,C*02:02, DPB1*05:01 C*15:02 OvCa 62 serous T3cN0G2R0 A*24:02,DQB1*03:01, DQB1*05:04, 12 ovarian A*31:01, DQA1*01:02, DQA1*03:01,carcinoma B*35:03, DRB1*01:01, DRB1*04:01, B*49:01, DRB4*01:01,DPB1*02:01, C*07:01, DPB1*05:01 C*12:03 OvCa 62 serous T1cN1G3R0 A*02,B*35, DQB1*04, DQB1*06, 13 ovarian B*40, C*03, DRB1*08, DRB1*13carcinoma C*04 OvCa 75 serous T3cN0G3R0 A*11:01, DQB1*03:01, DQA1*05:01,15 ovarian A*24:02, DRB1*11:01, DRB1*03:17, carcinoma B*07:02,DRB3*02:02, DPB1*03:01 B*55:01, C*03:03, C*07:02 OvCa 45 serousT3bN1G3R0 A*02, B*40, DQB1*06, DRB1*08, 16 ovarian B*44, C*03, DRB1*13,DRB1*14, DRB3 carcinoma C*05 OvCa 29 serous T3aN1G3R0 A*01, A*03DQB1*02, DQB1*03, 23 ovarian B*08, B*35, DRB1*03, DRB1*12, DRB3carcinoma C*04, C*07 OvCa 66 serous T2bN0G3R0 A*01:01, DQB1*05:01,DQB1*06:01, 28 ovarian A*02:01, DQA1*01:01, DQA1*03:01 carcinoma B*27.05DRB1*01:03, DRB1*15:02, B*52:01, DRB5*01:02, DPB1*04:01 C*01:02, C*02:02OvCa 45 serous T3cN1G3R1 A*25:01, DQB1*06:02, DQA1*01:02, 39 ovarianA*31:01, DRB1*15:01, DRB1*16:09, carcinoma B*07.02, DRB5*01:01,DRB5*01:11, B*18:01, DPB1*04:01, DPB1*04:02 C*12:03, C*07:02 OvCa 66serous T3cN0G3R1 A*02, A*24, DQB1*03, DQ7, DRB1*11, 41 and B*18, B*51,DRB3 endometrial C*02, C*12 ovarian carcinoma OvCa 61 serous T3cN1G3R2A*02, A*32, DQB1*03, DQB1*05, DQ9, 43 ovarian B*18, B*35, DRB1*01,DRB1*07, DRB4 carcinoma C*04, C*07 OvCa 63 mixed T1cN0G3R0 A*01, A*23,DQB1*02, DRB1*03, 45 differentiated B*08, B*44, DRB1*07, DRB3, DRB4(mostly C*04, C*07 endometroid) ovarian carcinoma OvCa 71 serousT3cN1G3R0 A*02:01, DQB1*03:02, DQB1*03:04, 48 ovarian A*25:01,DQA1*03:01, DRB1*04:01, carcinoma B*15.01, DRB1*13:03, DRB3*01:01,B*41:02, DRB4*01:01, DPB1*02:01 C*03:04, C*17:01 OvCa 48 serousT3bN1G3R0 A*02, A*03, DQB1*02, DQB1*03, DQ7, 53 ovarian B*27, B*35,DRB1*03, DRB1*11, DRB3 carcinoma C*02, C*04 OvCa 66 serous T3cN1M1G3R2A*02:01, DQB1*05:01, DQB1*05:03, 54 ovarian A*11:01, DQA1*01:01,DRB1*01:03, carcinoma B*35:01, DRB1*14:01, DRB3*02:02, B*35:03,DPB1*04:01, DPB1*02:01 C*04:01, C*12:03 OvCa 58 endometriod T1cN0G1R0A*25, A*32, DQB1*05, DQB1*06, 57 ovarian B*15, B*18, DRB1*01, DRB1*15,DRB5 carcinoma C*03, C*12 OvCa 74 serous T3cN1G3R1 A*02, A*03, DQB1*05,DRB1*01 58 ovarian B*35, C*03, carcinoma C*04 OvCa 47 serous T3cN1G3R2A*03, A*30, DQB1*02, DRB1*07, DRB4 59 ovarian B*13, C*06 carcinoma OvCa50 serous T3cN1G3R1 A*24:02, DRB1*08:01, DRB1*13:01, 60 ovarian A*25:01,DQB1*04:02, DQB1*06:03, carcinoma B*13:02, DQA1*04:01, DQA1*01:03,B*18:01, DPB1*02:01, DPB1*03:01 C*12:03, C*06:02 OvCa 56 serousT3cN1G3R1 A*01, A*25, DQB1*02, DRB1*03, DRB3 64 ovarian B*08, C*07carcinoma OvCa 55 serous T3cN1M1G3R1 A*01, A*24, DQB1*03, DQB1*05, 65ovarian B*15, B*35, DRB1*10, DRB1*11, DRB3 carcinoma C*04, C*14 OvCa 73serous T2bN0G3R0 A*11:01, DRB1*03, DRB*0701, 66 ovarian A*29:02,DRB3*0202, DRB4*0101, carcinoma B*18:01, DQB1*02:01, DQB1*02:02,B*44:03, DQA1*02:01, DQA1*05:01, C*05:01, DPB1*02:02, DPB1*03:01 C*16:01OvCa 69 serous T3cN1G3R1 A*02:01, DRB1*10:01, DRB1*04:01, 68 ovarianA*01:01, DRB4*04:01, DQB1*05:01, carcinoma B*44.02, DQB1*03:01,DQA1*01:01, B*37:01, DPB1*04:01 C*06:02, C*05:01 OvCa 68 serousT3cN0G1R1 n/a n/a 69 ovarian carcinoma OvCa 48 serous T3cN1M1G1R1 A*01,A*02, DQB1*03, DQB1*05, 70 ovarian B*07, C*07, DRB1*09, DRB1*14, DRB3,carcinoma DRB4 OvCa 53 serous T3bN1G3R0 A*03:01, DRB1*01:01, DRB1*03:01,72 ovarian A*01:01, DRB3*01:01, DQB1*05:01, carcinoma B*08.01,DQB1*02:01, DQA1*01:01, B*07:02, DPB1*04:01 C*07:02, C*07:01 OvCa 69serous T3cN1G3R0 A*01:01, DRB1*03:01, DRB1*03:42, 73 ovarian B*08:01,DRB3*01:01, DRB3*01:14, carcinoma C*07:01 DQB1*02:01, DQA1*05:01,DPB1*04:01 OvCa 79 endometrial T3bNxG1R1 A*02:01, DRB1*11:04,DRB1*07:01, 74 ovarian B*18:01, DRB3*02:02, DRB4*01:01, carcinomaB*51:01, DQB1*03:01, DQB1*02:02, C*07:02, DQA1*02:01, DQA1*05:01,C*15:02 DPB1*04:02, DPB1*02:01 OvCa 57 endometrial T2bN0G2R0 A*01:01,DQB1*03:03, DQA1*02:01, 79 ovarian A*31:01, DRB1*07:01, DRB1*09:01,carcinoma B*08:01, DRB4*01:01, DPB1*13:01, B*51:01, DPB1*02:01 C*07:01,C*15:02 OvCa 93 serous T3cNxG3R2 A*25:01, DRB1*01:01, DRB1*12:01, 80ovarian A*32:01, DRB3*02:02, DQB1*03:01, carcinoma B*18:01, DQB1*05:01,DQA1*01:01, B*39:01, DQA1*05:01, DPB1*04:01 C*12:03 OvCa 78 serousT3cNxG3R2 A*02:01, DRB1*04:02, DRBB1*11:01, 81 ovarian B*45:01,DRB4*01:01, DRB3*02:02, carcinoma B*56:01, DQB1*03:01, DQB1*03:02C*07:02, C*01:02 OvCa 48 serous T3cN1G3R0 A*01:01, DRB1*04:02,DRB1*03:01, 82 ovarian A*03:01, DRB4*01:01, DRB3*01:01, carcinomaB*08:01, DQB1*02:01, DQB1*03:02, B*38:01, DQA1*03:01, DQA1*05:01,C*07:01, DPB1*04:01, DPB1*13:01 C*12:03 OvCa 50 serous T1cN0G2R0 A*02,A*11, DQB1*03, DQB1*05, 83 ovarian B*51, B*55, DRB1*09, DRB1*14, DRB3,carcinoma C*03, C*15 DRB4 OvCa 70 serous T3cN1G3R1 A*02:01, DRB1*15:01,DRB5*01:01, 84 ovarian B*07:02, DQB1*06:02, DQA1*01:02, carcinomaB*44:02, DPB1*04:01, DPB1*04:02 C*07:02, C*05:01

For MHC class I the inventors could identify 22,920 unique peptides(mean 1,263/sample) emanating from 9,136 different source proteins (mean1,239/sample) reaching >90% of the estimated maximal attainable coverage(see FIG. 7A).

Example 2, Identification of Top Cancer Associated HLA Ligands

Aiming to extract the most specific HLA ligands for EOC from this vastcatalogue of data the inventors compared the HLA ligand source proteinswith an in-house database of benign sources (“HLA benign ligandomedatabase”) consisting of samples from PBMCs (n=30), bone marrow (n=10),liver (n=15), colon (n=12), ovary (n=4) and kidney (n=16). The HLAbenign ligandome database contains 31,032 peptides representing 10,012source proteins and was established using blood or bone marrow fromhealthy donors as well as histopathologically evaluated normal tissues,all analyzed with exactly the same pipeline as used for EOCs. Forcomparative profiling “one hit wonders” (i.e. peptides only presented onone source with low PSM count) were removed from both datasets toaccommodate for false positive hits. Comparative analysis of the tworespective datasets (see FIG. 2A) revealed 379 MHC class I sourceproteins to be presented exclusively by EOC in at least three of thetested patients, highlighting an EOC specific HLA peptide repertoire.The TOP100 EOC specific source proteins ranked according to theirfrequency of presentation are visualized in FIG. 2B. The most importantEOC specific HLA ligand source protein yielded by this analysis wasmucin 16 (MUC16) also known as cancer antigen 125 (CA-125). Overall morethan 80 different MUC16 derived HLA ligands (see Table 8) were presentedin nearly 80% of patients (26/34).

TABLE 8 Sequence ID No. Sources HLA AHSKITTAM 3 OvCa 80 B*39:01AVKTETSTSER 4 OvCa 12, OvCa 79 A*31:01 AVTNVRTSI 5 Ovca 59, OvCa 60 B*13DALTPLVTI 6 OvCa 74 B*51:01 DALVLKTV 7 OvCa 41, OvCa 74, OvCa 79, B*51OvCa 83 DPYKATSAV 8 OvCa 10, OvCa 41, OvCa 69 B*51 OvCa 74, OvCa 79,OvCa 83 EPETTTSFITY 9 OvCa 65 B*35 ERSPVIQTL 10 OvCa 80 B*39:01ETILTFHAF 11 OvCa 48, OvCa 64, OvCa 80 A*25 EVISSRGTSM 12 OvCa 48, OvCa60, OvCa 64, A*25 OvCa 80 EVITSSRTTI 13 OvCa 60, Ovca 64 A*25 EVTSSGRTSI14 OvCa 60, Ovca 64, OvCa 80 A*25 FPEKTTHSF 15 OvCa 65 B*35 FPHSEETTTM16 OvCa 13, OvCa 65 B*35 FPHSEITTL 17 OvCa 12, OvCa 13, OvCa 53 B*35FQRQGQTAL 18 OvCa 48 B*15:01 GDVPRPSSL 19 OvCa 72 B*08:01 GHESHSPAL 20OvCa 80 B*39:01 GHTTVSTSM 21 OvCa 80 B*39:01 GTHSPVTQR 22 OvCa 39, OvCa79 A*31:01 GTSGTPVSK 23 OvCa 83 A*11 HPDPQSPGL 24 OvCa 65 B*35 IITEVITRL547 OvCa 83 A*02 IPRVFTSSI 25 OvCa 41, OvCa 74 B*51 ISDEVVTRL 26 OvCa 16C*05 ISIGTIPRI 27 OvCa 65 B*15:17 ISKEDVTSI 28 OvCa 65 B*15:17 ITETSAVLY29 OvCa 65 A*01 ITRLPTSSI 30 OvCa 65 B*15:17 KDTAHTEAM 31 OvCa 68B*44:02 KEDSTALVM 32 OvCa 16 B*40/B*4 KEVTSSSSVL 33 OvCa 16, OvCa 70B*40/ B*44/? KMISAIPTL 548 OvCa 81, OvCa 83 A*02 LPHSEITTL 34 OvCa 12,OvCa 13 B*35 LTISTHKTI 35 OvCa 65 B*15:17 LTKSEERTI 36 OvCa 65 B*15:17QFITSTNTF 1 OvCa 60 A*24:02 RDSLYVNGF 37 OvCa 68 B*44:02 RETSTSQKI 38OvCa 60 B*18:01 RSSGVTFSR 39 OvCa 79 A*31:01 SAFESHSTV 40 OvCa 41, OvCa74, OvCa 79, B*51 OvCa 83 SATERSASL 41 OvCa 13, OvCa 16, OvCa 70 C*03/?SENSETTAL 42 OvCa 16, OvCa 70 B*40/ B*44/? SEQRTSPSL 43 OvCa 70 n.a.SESPSTIKL 44 OvCa 13, OvCa 70 B*40/? SPAGEAHSL 45 OvCa 72, OvCa 81, OvCa84 B*07/B*6 SPAGEAHSLLA 46 OvCa 81 B*56:01 SPHPVSTTF 47 OvCa 84 B*07:02SPHPVTALL 48 OvCa 9, OvCa 72, OvCa 84 B*07:02 SPLFQRSSL 49 Ovca 72B*0702 SPQNLRNTL 50 OvCa 23, OvCa 72, OvCa 84 B *35/ B*07:02SPRLNTQGNTAL 51 OvCa 72, Ovca 84 B*07:02 SPSEAITRL 52 Ovca 84 B*07:02SPSKAFASL 53 OvCa 9, OvCa 23, OvCa 39, B*35/ OvCa 69, OvCa 72, OvCa 84B*07:02 SPSSPTPKV 54 OvCa 72 B*07:02 SPSSQAPVL 55 OvCa 84 B*07:02SQGFSHSQM 56 OvCa 48 B*15:01 SRTEVISSR 57 OvCa 53 B*27 SSAVSTTTI 58 OvCa65 B*15:17 SSPLRVTSL 59 OvCa 69 n.a. STASSSLSK 60 OvCa 83 A*11 STETSTVLY2 OvCa 64, OvCa 65, OvCa 68 A*01 STQRVTTSM 61 OvCa 72 n.a. STSQEIHSATK62 OvCa 83 A*11 SVLADLVTTK 63 OvCa 72 A*03:01 SVPDILSTSW 64 OvCa 60A*24:02 TAGPTTHQF 65 OvCa 58 C*03 TEISSSRTSI 66 OvCa 12 B*49:01TENTGKEKL 67 OvCa 16 B*40/B*44 TETEAIHVF 68 OvCa 41, OvCa 80 B*18TEVSRTEVI 69 OvCa 12 B*49:01 TExVLQGLL 70 OvCa 16, OvCa 66, OvCa 70B*40/ B*44/? TPGGTRQSL 71 OvCa 9, OvCa 23, OvCa 39, B*07:02/ OvCa 72,OvCa 84 B*35 TPGNRAISL 72 OvCa 23, OvCa 72, OvCa 84 B*07:02/ B*35TPNSRGETSL 73 OvCa 72 B*07:02 TSGPVTEKY 74 OvCa 58 B*35 TSPAGEAHSL 75OvCa 81 n.a. TTLPESRPS 324 OvCa 70 n.a. TYSEKTTLF 549 OvCa 12, OvCa 41,OvCa 60, A*24 OvCa 65 VHESHSSVL 76 OvCa 80 B*39:01 VPRSAATTL 77 OvCa 23,OvCa 72, OvCa 84 B*07:02/ B*35 VTSAPGRSI 78 OvCa 65 B*15:17 VTSSSRTSI 79OvCa 65 B*15:17 YPDPSKASSAM 80 OvCa 65 B*35

Those data highlight the frequent processing and presentation of MUC16by a multitude of different HLA allotypes unparalleled by any other EOCspecific antigen and mirrored only by frequently (>95%) presentedhouse-keeping proteins such as beta actin (overall 149 differentpeptides identified). Among the TOP100 EOC specific source proteinsother well established tumor associated antigens like MUC1 or KLK10 aswell as antigens with well documented immune-evasive functions likeIndoleamine-2,3-dioxygenase (IDO1) or Galectin 1 (LGALS1) wereidentified.

Owing to the power of CD4 T cells in supporting or driving an anti-tumorimmune response the inventors used the same approach to further analyzeMHC class II presented peptides in EOC (n=22) yielding 9,162 peptides(mean 598/sample) representing 2,330 source protein (mean 319/sample)reaching >80% of attainable coverage (see FIG. 7B). The HLA benignligand dataset for MHC class II contained 7,267 peptides representing1,719 source proteins derived from bone marrow (n=5), PBMCs (n=13),colon (n=2), liver (n=7) and kidney (n=17). Analysis of the TOP100 MHCclass II presented antigens revealed a more heterogeneous and complexpicture (FIG. 2C). Notably, MHC presented peptides of mesothelin (MSLN)an established ligand of MUC16, could be identified in nearly 50% ofpatients (10/22; FIG. 2D). MUC16 itself was not among the TOP100 classII antigens but respective ligands could nevertheless be detected infour patients.

Besides the TOP100 EOC specific HLA ligand source proteins, theinventors further looked for established cancer-testis and tumorassociated antigens that have been previously employed for clinicalapplication to verify their abundance (Her2neu, WT1, NY-ESO-1, hTert andp53). Although the inventors could identify HLA presented peptides forall antigens except for NY-ESO-1, none of them were exclusivelypresented on EOC (Table 9). The only ligands showing EOC specificpresentation, albeit with low frequency (3/34), were HLA class I ligands(but not HLA class II) from Her2neu.

TABLE 9 SEQ HLA Sources of ID Her2neu restriction presentation ERBB2(Receptor tyrosine-protein kinase erbB-2) 554 TYLPTNASLSF A*23/A*24 2xOvCa 153 MPNPEGRYTF B*35 1x OvCa 152 AARPAGATL B*07 1x OvCa 291AIKVLRENTSPKANKE HLA class II 1x OvCa 292 DPSPLQRYSEDPTVPLPS HLA classII 2x OvCa 293 DPSPLQRYSEDPTVPLPSE HLA class II 1x OvCa 294 ELVSEFSRMARDHLA class II 2x PBMCs 295 ELVSEFSRMARDPQ HLA class II 2x PBMCs, 1xKidney 296 IPVAIKVLRENTSPKANKE HLA class II 1x OvCa 297 RRLLQETELVEPLTPSHLA class II 2x Liver 298 SPQPEYVNQPDVRPQPP HLA class II 1x OvCa 291VKPDLSYMPIWKFPDE HLA class II 1x OvCa WT-1 Wilms tumor protein 558RMFPNAPYL A*02 8x PBMCs, 1x Liver 557 QRNMTKLQL B*13 2x OvCa, 1x Liver,1x PBMCs 555 GVFRGIQDV B*13 2x OvCa 550 ALLPAVPSL A*02 1x OvCa hTertTelomerase reverse transcriptase 556 LMSVYVVEL A*02 2xPBMCs p53 Cellulartumor antigen p53 552 RPILTIITL B*07 4x PBMCs, 2x Liver, 2x Kidney, 3xOvCa 553 TYSPALNKMF A*24 1x PBMCs, 1x Liver, 2x OvCa 551 GRNSFEVRV B*271x PBMC, 1x Liver, 1x Kidney, 1x OvCa

Example 3: Cellular Origin of EOC Associated HLA Presented Peptides

Since EOCs embody not only cancer cells but rather represent aheterogeneous mixture of different cell types the inventors asked,whether the MHC class I TOP100 antigens were indeed originally presentedby cancer cells. For this purpose the inventors digested EOCs andseparated CD45⁺ leukocytes, EpCam⁺ tumor cells as well as stroma cellsnegative for the two markers (for enrichment efficiencies see Table 10)and subsequently the inventors performed HLA ligandomics individuallyfor each of the subsets.

TABLE 10 Cell enrichment efficiencies: Percentage of cells are given ineach fraction before (PreSort) and after MACSorting PreSort CD45⁺fraction EpCam⁺ fraction EpCam⁻ fraction Ovca CD45⁺ EpCam⁺ ViabilityCD45⁺ EpCam⁺ Viability CD45⁺ EpCam⁺ Viability CD45⁺ EpCam⁺ Viability 8474.7 18.3 80.2 93.5 6.2 71.6 10.7 85.7 88.2 4.5 22.1 64.0 73 23.1 12.381.2 95.7 1.7 77.2 3.4 73.3 87.6 1.7 3.2 87.4 70 76.2 8.83 78.9 96 1.382.7 3.4 94 66.4 3.1 4.5 65.4 60 77.4 5.2 92.3 94.8 1.7 90.2 5.2 79.788.7 3.8 10.7 89.5 57 31.9 50.5 94.1 93.6 5.0 90.6 1.4 95.3 96.7 0.8 7.295.3

The inventors used label free quantification to determine the source ofeach identified HLA ligand in a total of 5 EOCs (for a representativeexample see FIGS. 3A and 3B). As expected, MUC16 derived HLA ligands,identified on (4/5) EOC samples, were always found to be overrepresentedon enriched cancer cells with a median 5 fold overrepresentation (range1.8-135 fold) dependent on the efficiency of the enrichment. The sameheld true for several other frequently presented TOP100 antigens likeDDR1, SOX9, CRABP1/2, EYA2, LAMC2, MUC1 or KLK10. However a number ofother antigens especially those known to be upregulated by interferonsuch as toll like receptors (TLR3, TLR7) or 2′-5′-oligoadenylatesynthase-like protein synthase (OASL) could not be unambiguously shownto be presented by tumor cells but rather displayed strongoverrepresentation on CD45+ leukocytes and/or stroma cells. Apart fromtumor associated antigens the inventors also recognized ligands fromsource proteins with cell type specific expression. For example ligandsderived from CD8, CD132 or lymphocyte specific protein 1 (LSP1) werefound highly overrepresented on CD45+ cells and van Willebrand factor(vWF) most likely expressed by endothelial cells in the stroma was foundhighly overrepresented within the stromal subset emphasizing thestrength of this cell type specific approach.

Example 4: Immunogenicity Analysis of MUC16 Derived Ligands

For the applicability of peptide vaccines immunogenicity is a majorimperative. In order to evaluate the immunogenic potential of theidentified HLA ligands the inventors used a T-cell priming protocolinvolving artificial antigen presenting cells and T cells isolated fromblood of healthy donors. The results of this analysis for the number oneEOC associated antigen MUC16 are presented in Table 11. Among 23different peptides tested so far, 18 were shown to be immunogenic in atleast 1/3 donors. This nearly 80% recognition rate verifies the presenceof naïve MUC16 recognizing T cells in the human population. Similarresults have been obtained for other TOP100 antigens (e.g. IDO1,LGALS1).

TABLE 11 Immunogenicity analysis of EOC presented HLA ligands fromMUC16/CA-125 SEQ positive/ HLA Sequence ID tested donors A*01 STETSTVLY2 0/2 A*02 IITEVITRL 547  3/10 A*02 KMISAIPTL 548 4/6 A*03 SVLADLVTTK 630/1 A*11 STSQEIHSATK 62 2/6 A*11 GTSGTPVSK 23 0/5 A*24 TYSEKTLLF 549 2/2A*24 AVTNVRTSI 5 1/3 A*25 ETILTFHAF 11 2/2 A*25 EVITSSRTTI 13 1/1 A*25EVTSSGRTSI 14 2/3 A*25 EVISSRGTSM 12 1/3 B*07 SPHPVTALL 48 0/1 B*07SPQNLRNTL 50 1/1 B*07 LPHSEITTL 34 0/2 B*07 SPSKAFASL 53 2/2 B*07VPRSAATTL 77 1/2 B*07 TPGNRAISL 72 2/2 B*15 SQGFSHSQM 56 4/5 B*15FQRQGQTAL 18 1/6 B*27 ERSPVIQTL 10 1/2 B*51 DALVLKTV 7 1/3 B*51DPYKATSAV 8 3/3 8/10 allotypes 18/23 HLA ligands 34/73

Example 5: Biomarkers for HLA Ligand Presentation

Antigen specific cancer immunotherapy (e.g. peptide vaccination,adoptive T-cell transfer) requires a stringent selection of candidateantigens within a short timeframe. HLA ligandome analysis however, isnot always possible due to the lack of appropriate material. A feasiblealternative would be the use of biomarkers to predict the presence ofHLA ligands on the tumor cells. In order to evaluate whether, proteinexpression analyzed by immunohistochemistry (immunoreactivity score,IRS) could serve as a surrogate marker for HLA ligand presentation, theinventors analyzed the TOP100 MHC class I antigens MUC16 and IDO1 aswell as the TOP100 MHC class II antigen MSLN by immunohistochemistry andcorrelated the staining intensity (FIG. 4A) to the presence or absenceof HLA ligands on the same tumors. For both MUC16 and MSLN, stainingscores were significantly higher on tumors, which presented HLA ligandsof respective source proteins (FIG. 4C). The same was true for CA-125serum levels determined at the day of surgery (FIG. 4D), indicating thatthese parameters could be used for a proper selection of candidateantigens for peptide vaccination. In contrast, IDO1 did not show asignificant association with ligand presentation.

Example 6: Prognostic Relevance of the MUC16/MSLN Axis

Because of their importance as targets for immunotherapy the inventorswanted to assess whether MSLN and MUC16 are also of prognostic relevancein a patients similar to our immunopeptidome collective. For thispurpose the inventors analyzed the expression of both antigens as wellas the extent of T-cell infiltration by immunohistochemistry in a tissuemicroarray (TMA) of high grade serous ovarian cancers (FIGO stageII-Ill). In order to avoid prognostically relevant confounders theinventors restricted our analysis to 71 patients with optimally debulkedcancers (residual mass below <1 cm).

While the inventors did not observe any prognostic effect for MUC16staining, strong MSLN staining was associated with a notable borderlinesignificant (p=0.0572) decrease of median overall survival from 50 to 28months (FIG. 5A). Despite their different prognostic relevance, stainingscores for MUC16 and MSLN showed a direct and highly significantcorrelation (Spearman correlation coefficient r=0.5237; 95%c.i.=0.3159-0.6835, two tailed significance p<0.001).

For the evaluation of T-cell infiltration the inventors assessed thenumber of CD3 T cells in the intraepithelial compartment of the tumor(CD3E) and the fibrovascular stroma (CD3S) separately. Notably only thenumber of intraepithelial T cells showed a significant (p<0.0063)prognostic impact, whereas infiltration of the surrounding stroma alonehad no prognostic relevance (FIG. 5B). Only in a subgroup analysiscombining MSLN and CD3 staining a significant prognostic benefit fortumors with low MSLN and high T-cell infiltration could be observed(FIG. 5C) for both CD3E (p<0.001) and CD3S (p<0.0049). Most strikingly,the combination of high intratumoral T-cell infiltration (CD3E) and lowMSLN staining defined a subset of long term cancer survivors (10/11patients with confirmed survival beyond 3 years).

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The invention claimed is:
 1. A method of eliciting an immune response ina patient who has cancer, comprising administering to said patient acomposition comprising a population of activated T cells that killcancer cells in the patient that present a peptide, wherein said peptideconsists of the amino acid sequence of SEQ ID NO: 98, wherein saidcancer is selected from the group consisting of ovarian cancer,non-small cell lung cancer, small cell lung cancer, kidney cancer, braincancer, colon or rectum cancer, stomach cancer, liver cancer, pancreaticcancer, prostate cancer, leukemia, breast cancer, Merkel cell carcinoma,melanoma, esophageal cancer, urinary bladder cancer, uterine cancer,gallbladder cancer, and bile duct cancer.
 2. The method of claim 1,wherein the T cells are autologous to the patient.
 3. The method ofclaim 1, wherein the T cells are obtained from a healthy donor.
 4. Themethod of claim 1, wherein the T cells are derived from tumorinfiltrating lymphocytes or peripheral blood mononuclear cells.
 5. Themethod of claim 1, further comprising expanding T cells in vitro.
 6. Themethod of claim 1, wherein the peptide is in a complex with an MHCmolecule.
 7. The method of claim 1, wherein the composition furthercomprises an adjuvant.
 8. The method of claim 7, wherein the adjuvant isselected from the group consisting of anti-CD40 antibody, imiquimod,resiquimod, GM-CSF, cyclophosphamide, Sunitinib, bevacizumab,interferon-alpha, CpG oligonucleotides and derivatives, poly-(I:C) andderivatives, RNA, sildenafil, particulate formulations with poly(lactideco-glycolide) (PLG), virosomes, interleukin (IL)-1, IL-2, IL-4, IL-7,IL-12, IL-13, IL-15, IL-21, and IL-23.
 9. The method of claim 1, whereinthe activated T cells are cytotoxic T cells produced by contacting Tcells, in vitro, with an antigen presenting cell that expresses thepeptide in a complex with an MHC class I molecule on the surface of theantigen presenting cell, for a period of time sufficient to activatesaid T cell specifically against the peptide.
 10. The method of claim 9,wherein the antigen presenting cell is infected with a recombinant virusexpressing the peptide.
 11. The method of claim 10, wherein the antigenpresenting cell is a dendritic cell or a macrophage.
 12. The method ofclaim 9, further comprising stimulating the activated T cells in thepresence of an anti-CD28 antibody and IL-12 to clonally expand the Tcells.
 13. The method of claim 1, wherein the population of activated Tcells comprises CD8-positive cells.
 14. The method of claim 1, whereinthe cancer is ovarian cancer.
 15. The method of claim 7, wherein theadjuvant comprises IL-2.
 16. The method of claim 7, wherein the adjuvantcomprises IL-7.
 17. The method of claim 7, wherein the adjuvantcomprises IL-15.
 18. The method of claim 7, wherein the adjuvantcomprises IL-21.
 19. A method of eliciting an immune response in apatient who has ovarian cancer, non-small cell lung cancer, small celllung cancer, kidney cancer, brain cancer, colon or rectum cancer,stomach cancer, liver cancer, pancreatic cancer, prostate cancer,leukemia, breast cancer, Merkel cell carcinoma, melanoma, esophagealcancer, urinary bladder cancer, uterine cancer, gallbladder cancer,and/or bile duct cancer, comprising administering to said patient acomposition comprising a peptide in the form of a pharmaceuticallyacceptable salt, wherein said peptide consists of the amino acidsequence of SEQ ID NO: 98, thereby inducing a T cell response to theovarian cancer, non-small cell lung cancer, small cell lung cancer,kidney cancer, brain cancer, colon or rectum cancer, stomach cancer,liver cancer, pancreatic cancer, prostate cancer, leukemia, breastcancer, Merkel cell carcinoma, melanoma, esophageal cancer, urinarybladder cancer, uterine cancer, gallbladder cancer, and/or bile ductcancer.
 20. The method of claim 19, wherein the T cell response is acytotoxic T cell response.